Methods for preparing high throughput peptidomimetics, orally bioavailable drugs and compositions containing same

ABSTRACT

Provided herein are methods to generate and screen peptides that exhibit drug like stabilities in vitro and in vivo. By selecting for enzyme resistance, Applicants are able to derive peptides that are not only stable to a broad spectrum of proteases, but also stable to other drug processing enzymes such as cytochrome P450s. This approach provides a general method to the rapid development of highly stable peptides for therapeutic development and diagnosis. The peptides are further modified for oral bioavailability. The methods can be applied to similar peptides for the making of therapeutic compositions

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/342,347, filed Aug. 31, 2012, now U.S. Pat. No. 9,944,675, which is anational stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2012/053526, filed Aug. 31, 2012, which in turnclaims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Nos. 61/530,327, 61/530,352 and 61/530,372, each filed Sep.1, 2011, the contents each of which are incorporated herein by referenceinto the present disclosure.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under the NationalInstitutes of Health Grant No. R01 GM 60416. Accordingly, the U.S.Government has certain rights to the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 16, 2018, isnamed 064189-5120 SL.txt and is 40,597 bytes in size.

BACKGROUND

Improved understanding of cellular function as well as disease pathologyprovides many possible targets for developing new therapeutic anddiagnostic reagents. A general problem with turning this cellularknowledge into treatments is that many potential targets arefunctionally undruggable. Hopkins et al. (2002) Nat. Rev. Drug Discov.1:727. Therefore, it is not possible to develop small molecules that canmodulate many of the protein-protein interactions that control cellularfunction.

Monoclonal antibodies provide an important route to target proteinsurfaces and represent both a large and growing portion of approvedtherapeutics. Nelson et al. (2009) Nat. Biotech. 27:331. However,antibodies are expensive to produce and dose, difficult to administerand control quality, can produce neutralizing immune responses, andcannot be used to target intracellular proteins. Cho et al. (1996)Trends in Biotech. 14:153. The fact that such cumbersome moleculesdominate therapeutic protein-binding reagents demonstrates both theimportance of these reagents and our desperation at finding viablereplacements.

Peptides provide an attractive alternative route to developing this typeof ligand. Non-ribosomal proteins such as cyclosporine are peptidiccompounds that incorporate amino acids beyond the 20 common residuescoded for by DNA. These molecules are capable of modulatingintracellular protein-protein interactions (PPI), and exhibit oralbioavailability. However, there have been a limited number oftherapeutic molecules derived by searching natural products. Ho, S. etal. (1996) Clin. Immunol. And Immunopathol. 80:S40; Baumann et al.(1994) Protein Sci. 3:750; and De La Cruz et al. (1996) Biochem.35:14054.

Selection methods such as mRNA display and phage display have been shownto be capable to generating peptidic ligands able to bind to andmodulate PPI for a very large variety of protein interactions. Ja et al.(2004) Biochemistry 43:9265; Ja et al. (2006) ACS Chem. Biol. 1:570; andMillward et al. (2007) ACS Chem. Biol. 2:625. However, these peptidessuffer from poor bioavailability due in large part to proteolysis.

A more traditional approach to peptide medication and drug developmentrequires the generation of a lead molecule followed by extensivemedicinal experimentation. Present current strategies include helixstapling, peptoid synthesis, beta-peptide generation, and N-methylincorporation. Fiacco, S. V. and Roberts, R. W. (2008) ChemBio Chem9:2200; Walensky et al. (2004) Science 305:1466; Miller et al. (1994)Bioorganic & Medicinal Chem. Letters 4:2657; and Nguyen, J. T. et al.(1998) Science 282:2088. However these techniques are very laborintensive, often taking years, with no guarantee of success. Many timesthe resulting product will exhibit a loss in function, specificity, orboth.

Previous attempts to stabilize and enhance protease stability of apeptide include chemically scanning each of the 9 positions with anN-methyl analog. Four (4) of the 9 positions enhanced proteaseresistance indicating that a single N-methyl provided a window ofprotection around the scissile bond. The resulting protease resistancewas dramatic, ranging from 70 fold to over 1000 fold at the site ofcleavage. Unfortunately only one of the 9 substitutions retained thesame binding specificity as the parent molecule; 4 of the sequences lostfunction while 4 had altered specificity now binding the related proteinGα12 instead of the original target, gαi1. Interestingly combining thetwo best Gα12 binding modifications resulted in a total loss of function(Fiacco et al. (2008), supra).

Another approach involved making covalent cyclic mRNA display librariesthat had the unnatural amino acid N-methyl phenylalanine (NMF). Thatwork showed that cyclic peptides could have antibody like affinity andthat cyclization improved stability. However, the increase in proteaseresistance was modest (2.6 fold) and the resulting molecules lacked theunnatural amino acid (Millward et al. (2007) supra; Frankel et al.(2003) Chemistry & Biology 10:1043 and Gilmore et al., In Implementationand Redesign of Catalytic Function in Biopolymers; SpringerBerlin/Heidelberg (1999) 202:77). Additionally Phe is a large residueand may be compatible in only a few of the positions.

Thus, a need exists to devise a scheme that retains the binding functionof a natural peptide while dramatically improving its stability both invitro and in vivo, that can be applied to any target of interest,thereby overcoming the limitation of the helix stapling methodsdescribed by Walensky et al. (2004), supra., which can only be appliedto some helical structures.

A need also exists for new approaches to target unmet medical needs.Diabetes is an example. Diabetes can be divided into two categories.Type 1 diabetes is characterized by individuals with the inability tocreate insulin. This form of diabetes is a progressive diseasecharacterized by significant loss in pancreatic (3-cell mass leading toimpaired insulin secretion. Impaired insulin secretion results inhyperglycemia which can lead to serious health problems such asketoacidolysis if left untreated. Iyer, H., et al. (2010) Diabetes,Obesity and Metabolism 12:179. Type 2 is characterized by the inabilityto properly utilize insulin. Long term misregulation of blood sugarleads to an increased risk of heart failure, kidney disease, strokes,and limb amputation. Ford, E. S. Journal of Diabetes Ford, E. S. (Jul.8, 2011). In the United States, more than 90% of individuals withdiabetes have type 2 diabetes. WebMd 2011, available at the web address:diabetes.webmd.com/guide/type. Currently, diabetes is the seventhleading cause of death in the United States. Center for Disease Control2011, available at the web address:www.cdc.gov/diabetes/pubs/estimates11.html.

According to the World Health Organization as of 2011, approximately 220million people, or 3.2% of the world population, have diabetes. Thisnumber is expected to increase to 4.4% of the overall world populationby 2030. Goldberg, M., et al. (2003) Nat Rev Drug Discov. 2:289.Diabetes becomes more prevalent with age, effecting 18.3% (8.6 million)of Americans over 60 years old (Center for Disease Control 2011,available at the web address: www.cdc.gov/diabetes/pubs/estimates11.html).

There is a significant precedent for the development of peptides thatare capable of binding protein surfaces with high affinity andspecificity for the purpose of controlling protein-protein interactionsin vitro. Ja, W. et al. (2006) ACS Chem. Biol. 1:570; Karatan, E. et al.(2004) Chemistry & Biology 11:835; Fuh, G. et al. (2000) J. Biol. Chem.275:21486; and Stoop, A. A. and Craik, C. S. (2003) Nat Biotech 21:1063.However, their development into orally bioavailable therapeutics ortherapeutics able to reach intracellular targets remains a tremendouschallenge. This is due in part to the intrinsic difficulties these typesof ligands face in crossing lipid bilayers. Morris, M. C. et al. (23001)Nat Biotech 19:1173; Goldberg, M. and Gomez-Orellana, I. (2003) Nat RevDrug Discov. 2:289. There are examples pharmacologically relevantpeptidic ligands that are thought to passively diffuse across cellmembranes such as cyclosporine, amanitin, and phalloidin. Ho, S. et al.(1996) Clinical Immunology and Immunopathology 80:S40; Baumann, K. etal. (1994) Protein Sci. 3:750.; and De La Cruz, E. M. and Pollard, T. D.(1996) Biochemistry 35:14054.

Current biological treatments for diabetics typically center around theinjection of various insulin products. Sales of insulin and insulinanalogs resulted in over $15 billion in revenue in 2010. Currently,there is no FDA approved oral formulation for insulin. Developing anoral route to insulin administration would be a tremendous benefit tothose affected by the diabetes, not only because of convenience, butalso due to potential health benefits of this route of administration.After secretion from the pancreas, insulin travels to the liver beforedispersion to the rest of the body. Orally absorbed drugs follow thissame route; however drugs administered by injection do not. It ispostulated that an oral treatment would alleviate adverse side effectsof insulin administration such as weight gain and hypoglycemia. Funnell,M. M. (2006) Clinical Diabetes 24:154.

Effective treatment of diabetes also is hindered by low patientcompliance. Today, insulin is administered by self-injection. An orallybioavailable drug would enhance patient compliance. However, orallyadministered drugs, such as peptidic ligands, are hindered by the factsthat peptidic ligands must reach their targets to be effectivediagnostically or therapeutically. Additionally, intracellular proteinsmust cross the plasma membrane and bioavailable peptides must traversethe intestinal mucosa to reach their targets. This invention addressesthe limitations of the current state of the art.

SUMMARY

In one aspect, Applicants provide a method for selecting one or morenucleic acids from a library of nucleic acids that encode one or morestable and bioavailable peptide(s), comprising, or alternativelyconsisting essentially of, or yet further consisting of, selecting froma nucleic acid library one or more nucleic acids encoding stable andbioavailable peptide(s), wherein one or more members of the nucleic acidlibrary contain one or more unnatural amino acids and/or codon lackingnatural cognate tRNAs.

In another aspect, this invention provides a method for selecting one ormore more peptides that possess secondary structure. This methodcomprises, or alternatively consisting essentially of, or yet furtherconsist of, the steps of generating a library of peptides and selectingindividual sequences from the library by treating the library with oneor more proteases, thereby selected for one or more peptides withsecondary structure. In one aspect, the nucleic acid is an RNA and/orthe library is an RNA library. In a further aspect, the method furthercomprises supplementing the library with one or more tRNA comprising anamino acid to suppress the stop codon. The members of the mRNA libraryare linear and/or cyclic.

In another aspect, a method is provided for preparing one or morenucleic acids that encode peptide(s), comprising the steps of mutating alibrary of peptides selected for a pre-determined specificity toincorporate amino acids that impart stability; incorporating one or morestop codons; supplementing tRNA charged with the desired amino acid tosuppress the stop codon; cyclizing one or more of the individualsequences of the nucleic library; selecting individual sequences of thelibrary for protease resistance, thereby selecting for one or morenucleic acids that encode one or more peptide(s). In one aspect, thenucleic acid library is a DNA library or a RNA library. In one aspect,the method further comprises translating the one or more nucleic acidsto a peptide. In a further aspect, the method further comprisesconjugating the peptide to a biotin molecule or biotin analog through alinkage. Non-limiting examples of biotin molecules or biotin analogs areone or more of a reducible biotin molecule; a biotin on the side chainof lysine of the peptide; a biotin linked to the peptide through anamide linkage or an ester linkage, a biotin linked to the peptidethrough a thioester linkage or an ester linkage. In a further aspect ofthe method, the nucleic acid is isolated. The nucleic acid is optionallytranslated into the encoded peptide or protein and further isolated. Theisolated molecules are further provided by this invention.

Peptides generally have poor stability and bioavailability in vivo.Here, Applicants provide methods to prepare one or more RNA that encodeone or more stable and bioavailable peptide(s) by selecting individualsequences from an mRNA library, wherein the library contains nucleicacids encoding peptides selected for pre-determined specificity andstability, thereby selecting for one or more RNA that encode one or morestable and bioavailable peptide(s). In one aspect, the one or moremembers of the RNA library have one or more stop codons. In anotheraspect, the methods further comprise supplementing the library with oneor more tRNA charged with the desired amino acid(s) to suppress the stopcodon. In another aspect, one or more members of the mRNA library islinear or cyclic In one aspect, one or more members of the mRNA libraryhave a single amino acid that confers stability, e.g., an N-methyl aminoacid, an AIB, a D-amino acid or a beta (B) amino acid, or one thatconfers unique functionality on a side chain in such as manner as toderive functional peptides that exhibit drug like stabilities in vitroand in vivo. By selecting for example, protease resistance, e.g.,resistance to a caspase, an endopeptidase, an aminopeptidase, trypsin,thrombin, pepsin, chymotrypsin and proteinase K resistance, Applicantsare able to derive peptides that are not only stable to a broad spectrumof proteases, but also stable to other drug processing enzymes such ascytochrome P450s. Additionally, simple modifications provide significantincreases to in vivo half-life, as well as significant oralbioavailability. This approach provides a general method to the rapiddevelopment of highly stable peptides for therapeutic development anddiagnosis.

In one aspect, on the DNA level, the library is designed to either codefor the wild type amino acid or an amino acid that confers stability onthe peptide, e.g., one or more of an N-methyl amino acid, an AIB, aD-amino acid or a beta (B) amino acid, or one that confers uniquefunctionality on a side chain. In doing so, other amino acids are codedfor as a byproduct. This provides library diversity. The DNA libraryoptionally also has a codon coding for methionine (Met) at the 5′end,and a codon encoding lysine (Lys) at the 3′ end. Typical mRNA displaydesign can be employed from that point on. The lysine also can be usedin cyclization. Chemically acylated (charged) tRNA with the N-methylamino acid is designed to suppress a stop codon within the sequence,e.g., the stop codon UAG. This is supplemented to a typical translationmixture (e.g., rabbit retulocyte lysate).

Extra selective pressure can be applied for stabilized peptides(peptides chemically linked to their encoding mRNA named fusions).Translated products are subject to protease degradation e.g., theimmobilized, purified proteases proteinase K, chymotrypsin,aminopeptidase, and trypsin. After proteolysis, proteases can be removedfrom fusions by filtration.

Additional selection pressure can be applied. For example, selection canbe performed against human cells from a cancer patient thatoverexpressed a protein such as Her-2. In this example, the target is ahuman protein with post-translational modifications relevant in thedisease state. This is a significant technological advance because themethod and the peptides selected by its use allows the targeting ofproteins that contain post-translational modifications in the diseasestate and proteins that are difficult to overexpress or purify.

For each of the above methods that require a selection, this inventionprovide several selection methods. Non-limited methods include selectingagainst a cell surface protein; selecting against mammalian cell cultureor tissue; selecting against a purified protein target; selectingagainst a purified protein target that is a SUPR target; selecting foran in vivo therapeutic utility such as one or more of: the ability toinhibit the growth or kill a cancer cell, the ability to inhibit or killa pre-cancerous cell or to bind to a cell expressing a desired targetreceptor.

In one aspect, the disclosure also provides an isolated RNA libraryprepared by the methods as described herein or an isolated peptidelibrary prepared by a method described above. In another aspect, thedisclosure provides an isolated peptide prepared by a method describedherein.

The libraries, peptides and nucleic acids can be combined with acarrier, such as a pharmaceutically acceptable carrier.

This disclosure also provides non-naturally occurring peptidescomprising amino acid sequences of the group:

-   -   1) MAVYVHYHK, wherein Position 1 is selected from Met,        norvaline, alanine, or norleucine; Position 2 is selected from        Ala or V, M, S, T, H, K, R, Q, N, L, V, or I; Position 3 is        selected from N-Methyl Norvaline, S, T, Q, N, H, P, I, V, L, Y,        F, or P; Position 4 is selected from Tyr, V, Y, F, Q, N, S, T,        or H; Position 5 is selected from N-Methyl Norvaline, Y, F, S,        T, E, D, M, A, or P; Position 6 is selected from His, V, Y, F,        Q, or N; Position 7 is selected from His, V, F, Y, V, I, or L;        Position 8 is selected from His or any amino acid; and Position        9 is selected from Lys or lysine derivatives, e.g., Orn (SEQ ID        NO: 1);

2) MFVQVYYHK, wherein Position 1 is selected from Met, norvaline,norleucine, or alanine; Position 2 is selected from Phe or any aminoacid; Position 3 is selected from N-methyl norvaline, Q, N, S, T, H, Y,F, or P; Position 4 is selected from Gln, Y, F, V, P, S, or T; Position5 is selected from N-methyl norvaline, Y, F, S, T, D, E, A, or M;Position 6 is selected from Tyr, F, or H; Position 7 is selected fromTyr, F, L, I, V, S, T, or V; Position 8 is selected from His, T, or S;Position 9 is selected from Lys or lysine derivatives, e.g., Orn (SEQ IDNO: 2);

3) MLHYVYVRK, wherein Position 1 is selected from Met, norvaline,norleucine, or lanine; Position 2 is selected from Leu, I, or V;Position 3 is selected from His, Y, or F; Position 4 is selected fromTyr or F; Position 5 is selected from N-methyl norvaline, S, T, D, E, A,M, or P; Position 6 is selected from Tyr, H, Q, N, L, I, V, or V;Position 7 is selected from N-methyl norvaline, F, Y, L, I, V, H, or P;Position 8 is selected from Arg or any amino acid; Position 9 isselected from Lys or lysine derivatives, e.g., Orn (SEQ ID NO: 3);

4) MVCVVLYDDK, wherein Position 1 is selected from Met, norvaline,norleucine, or alanine; Position 2 is selected from Val, I, or L;Position 3 is selected from Cys; Position 4 is selected from N-methylnorvaline, Y, F, P, D, E, or M; Position 5 is selected from N-methylnorvaline, Y, F, D, E, W, C, G, or P; Position 6 is selected from Leu,Y, F, V, V, I, P, or C; Position 7 is selected from Tyr, V, E, or D;Position 8 is selected from Asp, S, T, E, Y, F, A, P, or V; Position 9is selected from Asp, E, G, L, I, or V; Position 10 is selected from Lysor lysine derivatives, e.g., Orn (SEQ ID NO: 4);

5) MEVYEYVSLK, wherein Position 1 is selected from Met, norvaline,norleucine, or alanine; Position 2 is selected from Glu or any aminoacid; Position 3 is selected from N-methyl norvaline, P, D, E, F, Y, S,T, Q, or N; Position 4 is selected from Tyr, D, E, F, V, or P; Position5 is selected from Glu, D, Y, F, P, or V; Position 6 is selected fromTyr, F, L, V, I, P, or V; Position 7 is selected from N-methylnorvaline, F, L, V, I, P, or V; Position 8 is selected from Ser or anyother amino acid; Position 9 is selected from Leu or any other aminoacid; Position 10 is selected from Lys or lysine derivatives, e.g., Orn(SEQ ID NO: 5);

6) MNEYVLYVLK, wherein Position 1 is selected from Met, norvaline,norleucine, or alanine; Position 2 is selected from Asn or any aminoacid; Position 3 is selected from Glu, D, I, V, L, F, Y, P, or V;Position 4 is selected from Tyr, D, E, P, or V; Position 5 is selectedfrom N-methyl norvaline, D, E, F, Y, G, C, or P; Position 6 is selectedfrom Leu, Y, F, P, or V; Position 7 is selected from Tyr, F, V, I, or L;Position 8 is selected from N-methyl norvaline, S, T, Y, F, E, D, A, orP; Position 9 is selected from Leu, K, R, I, L, V, D, E, G, S, or T;Position 10 is selected from Lys or lysine derivatives, e.g., Orn (SEQID NO: 6);

-   -   and for each of the above, V is an N-methyl amino acid or any        modified amino acid that confers stabilization to the peptide.

Also disclosed are peptide conjugates containing the above-notednon-naturally occurring peptides, host cells and compositions containingone or more of these peptides, polynucleotides, conjugates and/or hostcells. Also disclosed are polynucleotides encoding the peptides, vectorsand host cells containing these, as well as compositions containing anyone or more of the peptides, peptide conjugates, host cells,polynucleotides, vectors, alone or in combination with each other. Anyof the above-noted can be formulated into a composition comprising acarrier such as a pharmaceutically acceptable carrier. In one aspect,when the peptide targets the Her-2 receptor, a composition containingthe peptide is useful to inhibit the growth of a breast cancer cell invitro or in vivo. In one aspect, the contacting is in vivo and atherapeutically effective amount of the composition is administered. Ina further aspect, the patient is a Her-2+ patient.

This disclosure also provides a simple, nontoxic peptidic modificationthat can be incorporated into peptides and proteins, in one aspect thepeptides described herein, to facilitate their delivery across cellularmembranes as well as facilitate oral uptake. In one aspect, the peptidescomprise N- or C-terminal biotinylation which was discovered to resultin enhancements of bioavailability up to two orders of magnitude. Thepeptides optionally also comprise a disulfide between the biotin and thepeptide to enhance delivery to the target. This modification allowed forthe efficient delivery of peptide cargo in mammalian cells, as well as asignificant increase in oral delivery of a stable peptide, e.g.,insulin.

Thus, in one aspect, the disclosure provides a peptide conjugate,comprising, or alternatively consisting essentially of, or yet furtherconsisting of a peptide linked at the N- and/or C-terminal to a biotinmolecule or biotin analog through a linker, which includes but is notlimited to a disulfide linkage, an ester linkage or a amide linkage. Inone aspect, the biotin is a reducible biotin molecule. Additionalexamples of possible biotin analogs are provided herein.

In a further aspect, the peptide is linked to biotin on the side chainof lysine of the peptide. The linkages provide peptides and proteinssuitable for oral deliver and that facility intracellular delivery ofthe peptide or protein.

This disclosure also provides a method for inhibiting the growth of abreast cancer cell, comprising contacting the cell with an effectiveamount of a non-naturally occurring peptide, or the conjugate asdescribed herein, or the composition as described herein. Thesecompositions can also be used to treat breast cancer in subject in needthereof. Further provides id a method for detecting a breast cancer cellin a subject comprising administering to the subject one or more of anon-naturally occurring peptide as described herein, the conjugate asdescribed herein, or the composition as described herein and thenscreening for the presence of the presence of any peptide bound to thebreast cancer cell in the subject. In one aspect, the administeredcomposition detectively labeled, e.g., with a fluorescent dye or a PETlabel. In one aspect, the human patient is HER2+ patient.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B are general schematics of an N-methyl cyclicmRNA-display. FIG. 1A shows the design of an N-methyl library fromcyclic GIBP (also identified as “GiBP” herein) (SEQ ID NOS 19 and 51-52,respectively, in order of appearance). Boxed amino acids were found inthe selected peptide. FIG. 1B shows a selection strategy includingproteolysis.

FIGS. 2A to 2C are exemplary Scanning Unnatural Protease Resistant“SUPR” peptides. FIG. 2A shows a selected peptide sequence (SEQ ID NO:53). FIG. 2B is the chemical structure of cycGIBP. FIG. 2C is thechemical structure of an exemplary SUPR peptide. Location of N-methylincorporation is illustrated in light gray scale, change in side-chainstructure found in a darker gray scale.

FIGS. 3A to 3D show that a SUPR peptide retained the selectivity ofcycGIBP. NeutrAvidin-agarose was pretreated with (FIG. 3A) C-terminallybiotinylated cycSUPR or (FIG. 3B) C-terminally biotinylated cycGIBPpulled down various radiolabeled Gα subunits. Binding is normalized toGαi1 binding. Percent values represent the average of three experiments,and error is given in SEM see FIG. 3C. NeutrAvidin-agarose waspretreated with C) C-terminally biotinylated cycSUPR peptide or FIG. 3D)C-terminally labeled cycGIBP and incubated with Gαi1 in either the GDPstate or the active GTP (GTPγS) state.

FIGS. 4A to 4F show that a selected SUPR peptide (SEQ ID NO: 7) isresistant to proteolysis. FIG. 4A shows the predicted chymotrypsindigestsiteswith arrows above the sequence. Actual digest sites determined byMALDI-TOF and illustrated below the sequence. FIG. 4B shows thehalf-life of linGIBP(▴), cycGIBP(▪), linSUPR(•), and cycSUPR(♦) forchymotrypsin digest. FIG. 4C shows the predicted proteinase k digestsites in red. Actual digest sites determined by MALDI-TOF andillustrated in blue (SEQ ID NO: 7). FIG. 4D shows the half-life oflinGIBP(▴), cycGIBP(▪), linSUPR(•), and cycSUPR(♦) for proteinase Kdigest. FIG. 4E shows human serum half-life of half-life of linGIBP(▴),cycGIBP(▪), linSUPR(•), and cycSUPR(♦). FIG. 4F shows half-lifestability to human liver microsomes for half-life of linGIBP(▴),cycGIBP(▪), linSUPR(•), and cycSUPR(♦).

FIGS. 5A and 5B show cycSUPR optimized both Km and Kcat. FIG. 5A is Vmaxfor chymotrypsin digestion of cycGIBP(•), linSUPR(♦), and cycSUPR(▪). Aclose up of linSUPR and cycSUPR is embedded in the Figure to show thedata in more detail. FIG. 5B is a Table showing values for Km and Vmaxas derived by processing the data through Graphpad Prism 5.0. Both Kmand Vmax are highly optimized for protease resistance in cycSUPR peptidewhen compared to cycGIBP.

FIGS. 6A and 6B illustrate in vivo stability of a SUPR peptide. FIG. 6Ashows the sequence of a SUPR peptide (SEQ ID NO: 54) with fluorescein onthe side chain of lysine. FA-SUPR peptide (SEQ ID NO: 55) has anadditional modification to include palmitoleic acid on the side chain ofa C-terminal lysine. FIG. 6B shows in vivo stability of cycGIBP(▴),linSUPR(▪), cycSUPR(♦), and FA-cycSUPR(•). Y-axis shows percent peptideintact (undigested).

FIGS. 7A and 7B show that biotinylation and palmitoleic acid conjugationenhances oral bioavailability. FIG. 7A shows percent oralbioavailability vs time after feeding mice biotinylated peptide by oralgavage. FIG. 7B shows percent oral bioavailability of cyclic peptide vsbiotinylated cyclic peptide versus palmitoleic acid cyclic peptide. InFIGS. 7A and 7B, data represents the average of two points and error isshown as standard deviation.

FIG. 8 illustrates the design of the Her-2 N-methyl scanning library.Previous work (Park, B., et al. (2000) Nat Biotech 18:194, Nakajima, H.,et al. (2008) Breast Cancer 15:65) identified and optimized the residuesof the monoclonal antibodies used in Her-2 binding. From that, librarieswere constructed using the residues highlighted in gray scale. Aflanking random amino acid was placed on either side of the sequence.Met to Lys cyclization was also incorporated. Millward, S. et al. (2007)ACS Chem. Biol., 2:625. Sequences disclosed as SEQ ID NOS 56-57,respectively in order of appearance.

FIG. 9 illustrates Her-2 N-methyl library design (9A) and the design ofN-methyl library from cyclic GIBP (9B). Boxed amino acids were found inthe winning peptide. V represents N-methyl norvaline (SEQ ID NOS 58, 60,59 and 61, respectively, in order of appearance).

FIG. 10 shows enhanced translation efficiency of N-methyl norvaline.N-methyl norvaline (N-MeNva) translates approximately twice asefficiently as N-methyl alanine (NMA) in the context of a MFFXFF (SEQ IDNO: 73), template where X is the N-methyl amino acid. The sidechain ofN-methyl norvaline is also an isostere for methionine, which is aprevalent amino acid found in protein-protein binding domains.

FIGS. 11A and 11B show quantitation of Her-2 expressed on SKBR-3 cells.FIG. 11A shows fluorescent labeled HRAP is binding to SKBR-3 cells.Growth media, trypsin solution, and nonfunctional peptide all showsignal at background. FIG. 11B shows that 200,000 cells bind toapproximately 1.5 pmoles of peptide.

FIGS. 12A and 12B show evolution of peptides from their parentalmolecules. FIG. 13A shows a peptide taken from the Herceptin antibodyloop was put in the context of a MX₈K cyclic peptide and randomized aspreviously illustrated in FIG. 1 The amino acid sequence is shown aboveits chemical structure (SEQ ID NOS 62-63, respectively, in order ofappearance). Insertion of N-methyl amino acids is highlighted in red,and other changes are shown in the chemical structure in blue. FIG. 12Bshows a peptide taken from the Omnitarg antibody loop that was put inthe context of a MX₇K cyclic peptide and randomized as previouslydescribed (SEQ ID NOS 64-65, respectively, in order of appearance). Theamino acid sequence is shown above its chemical structure. Insertion ofN-methyl amino acids is highlighted in red, and other changes are shownin the chemical structure in blue.

FIG. 13 shows the inhibition of cellular proliferation with peptide madeby the disclosed methods. Shown is the maximal effect on proliferationof Her-2 positive cancer cells (SKBR-3 cells, in blue). Percentinhibition calculated by 100−(% proliferation). HeLa cells are used as acontrol because they are not a Her-2 overexpressing line.

FIG. 14 shows selected peptides that show in vivo efficacy. Individualmouse curves are labeled. Mice were dosed at 7 mg/kg by intravenousadministration 3 times weekly.

FIG. 15 is a proposed schematic displaying the mechanism of biotinmediated peptide shuttling.

FIG. 16 shows the oral availability by PAMPA log Pe measurements. Stoop,A. A., et al. (2003) Nat Biotech 21:1063. Log Pe values associated with50% availability or above in green, 5-75% in yellow, and below 5% inred. A notable exception is cyclosporine which has a log Pe of between−6.6 and −6.3 but is 25% bioavailable. Reducible biotin-labeled peptidesdenoted by RB following their number.

FIGS. 17A to 17G show the biotin mediated transport of peptide acrossthe plasma membrane. Images are the fluorescent channel followed by thelight channel. FIG. 17A shows peptide sequences used incorporating abridging disulfide between the biotin conjugate and the peptide (SEQ IDNOS 66-69, respectively, in order of appearance). FIG. 17B shows 3T3cells incubated with peptide 1 overnight. FIG. 17C shows HeLa cellsincubated with peptide 1 overnight. FIG. 17D shows 3T3 cells incubatedwith peptide 2 overnight. FIG. 17E shows HeLa cells incubated withpeptide 2 overnight. FIG. 17F shows neurons incubated overnight withpeptide 3. FIG. 17G shows neurons incubated overnight with peptide 4.

FIGS. 18A to 18D show that biotin shuttling not dependent onendocytosis. 3T3 cells are incubated with peptide 1 and examined byconfocal using the fluorescein channel (FIG. 18A) or the rhotaminechannel (FIG. 18B). 3T3 cells have been treated with the endocytoticinhibitor dynasore prior to peptide administration and are examinedunder the fluorescein channel (FIG. 18C) or the rhotamine channel (FIG.18D).

FIG. 19 shows the quantification of peptide uptake via flow cytometry.Shown is a comparison of the uptake efficiency of peptide 1 via biotinshuttling vs. TAT and polyarginine delivery. As a control, peptide 1 wasused that had been reduced and desalted prior to administration. “Arg-9”is disclosed as SEQ ID NO: 70.

FIGS. 20A and 20B show biotinylation and palmitoleic acid conjugationenhances oral bioavailability. FIG. 20A shows percent oralbioavailability vs time after feeding mice biotinylated peptide by oralgavage. FIG. 20B shows percent oral bioavailability of cyclic peptide vsbiotinylated cyclic peptide vs palmitoleic acid cyclic peptide. In FIGS.20A and 20B, data represents the average of two points and error isshown as standard deviation.

FIGS. 21A to 21C show biotinylated insulin regulates blood sugar levelsin vivo. FIG. 21A shows the site of biotinylation is on the N-terminusof the B-chain of insulin as determined by chymotrypsin digest and massspec analysis. Sequences are disclosed as SEQ ID NOS 71-72,respectively, in order of appearance. FIG. 21B shows the insulinresponse from 4 mice having biotinylated insulin administered at 3.5nmol/kg intravenously. FIG. 21C shows the insulin response from 4 micehaving unmodified human insulin administered at a dose of 3.5 nmol/kgintravenously.

FIG. 22 shows oral bioavailability of biotinylated insulin. FIG. 22shows blood glucose levels in vivo when mice are administered phosphatebuffer (▪), insulin (▴) at 7 ng/Kg, or biotinylated insulin (♦) at 7ng/kg. Points represent the average response for four mice and error isin SEM.

FIG. 23 shows half-life of peptides digested in human serum. Peptide(n=3) were digested in 95% human serum at 37° C. PMP (▴), HMP(▪), andSUPR(♦) peptides are shown.

FIG. 24 shows the half-life of peptides processed by human livermicrosomes. Peptide (n=3) were digested in 95% human serum at 37° C. PMP(▪), HMP(▴), and SUPR(•) peptides are shown.

FIGS. 25A through 25D show a method to produce structured peptides asexplained in Experiment No. 4. FIG. 25A shows that linGIBP (the startingpeptide) has a CD profile indicating that it is entirely unstructured.FIG. 25B shows that cyclic SUPR peptide is highly structured, and seemsto be helical. FIG. 25C shows that removal of the N-methyl amino acidsresults in a loss of helicity. FIG. 25D shows that linear SUPR peptideis also helical.

FIGS. 26A through 26E show that SUPR peptides are structured. FIG. 26Ashows a SUPR peptide having a helical profile. FIG. 26B shows thatpeptide D, of the Her-2 binding peptide family, shows a distortedhelical structure. FIG. 26C shows that Peptide 7, of the Her-2 bindingpeptide family, shows a beta-turn structure. FIG. 26D shows that Peptide1, of the Her-2 binding peptide family, has a beta turn profile verysimilar to cyclosporine (FIG. 26E). −10,000 mean molar ellipticity unitscorrelates to approximately −2.7 mdeg units. Therefore not only arepeptide 1 and cyclosporine have a similar shape, but also their degreeof structure is very similar.

FIG. 27 shows the results of analysis of toxicity of peptides asanalyzed by mtt. Peptide (n=3) was administered at 100 μM for 36 hours.DMSO concentration was 1% by volume. Cells were analyzed using a Abnovamtt assay kit. Plotted are the average values, and error in standarddeviation.

FIG. 28 shows immunogenicity of cycGIBP and SUPR with Freund's partialadjuvant (n=3). Positive control is biotinylated mouse antibody.Negative control is biotin. cycGIBP in blue, and SUPR peptide in red.Plotted are the average values and standard deviation.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All nucleotide sequencesprovided herein are presented in the 5′ to 3′ direction. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods, devices, and materials are now described. Alltechnical and patent publications cited herein are incorporated hereinby reference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); and Herzenberg et al. eds (1996) Weir's Handbook ofExperimental Immunology.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate oralternatively by a variation of +/−15%, or alternatively 10% oralternatively 5% or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a polypeptide” includes a plurality ofpolypeptides, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

A “subject” of diagnosis or treatment is a cell or an animal such as amammal, or a human. Non-human animals subject to diagnosis or treatmentand are those subject to infections or animal models, for example,simians, murines, such as, rats, mice, chinchilla, canine, such as dogs,leporids, such as rabbits, livestock, sport animals, and pets.

The term “protein”, “peptide” and “polypeptide” are used interchangeablyand in their broadest sense to refer to a compound of two or moresubunit amino acids, amino acid analogs or peptidomimetics. The subunitsmay be linked by peptide bonds. In another embodiment, the subunit maybe linked by other bonds, e.g., ester, ether, etc. A protein or peptidemust contain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of this invention that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

The term “isolated” or “recombinant” as used herein with respect tonucleic acids, such as DNA or RNA, refers to molecules separated fromother DNAs or RNAs, respectively that are present in the natural sourceof the macromolecule as well as polypeptides. The term “isolated orrecombinant nucleic acid” is meant to include nucleic acid fragmentswhich are not naturally occurring as fragments and would not be found inthe natural state. The term “isolated” is also used herein to refer topolynucleotides, polypeptides and proteins that are isolated from othercellular proteins and is meant to encompass both purified andrecombinant polypeptides. In other embodiments, the term “isolated orrecombinant” means separated from constituents, cellular and otherwise,in which the cell, tissue, polynucleotide, peptide, polypeptide,protein, antibody or fragment(s) thereof, which are normally associatedin nature. For example, an isolated cell is a cell that is separatedfrom tissue or cells of dissimilar phenotype or genotype. An isolatedpolynucleotide is separated from the 3′ and 5′ contiguous nucleotideswith which it is normally associated in its native or naturalenvironment, e.g., on the chromosome. As is apparent to those of skillin the art, a non-naturally occurring polynucleotide, peptide,polypeptide, protein, antibody or fragment(s) thereof, does not require“isolation” to distinguish it from its naturally occurring counterpart.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) having a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. The alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in Current Protocols in MolecularBiology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table7.7.1. Preferably, default parameters are used for alignment. Apreferred alignment program is BLAST, using default parameters. Inparticular, preferred programs are BLASTN and BLASTP, using thefollowing default parameters: Genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST.Sequence identity and percent identity were determined by incorporatingthem into clustalW (available at the web address://align.genome.jp/,last accessed on Mar. 7, 2011.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, or alternatively less than 25% identity, withone of the sequences of the present invention.

“Homology” or “identity” or “similarity” can also refer to two nucleicacid molecules that hybridize under stringent conditions.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: incubationtemperatures of about 25° C. to about 37° C.; hybridization bufferconcentrations of about 6×SSC to about 10×SSC; formamide concentrationsof about 0% to about 25%; and wash solutions from about 4×SSC to about8×SSC. Examples of moderate hybridization conditions include: incubationtemperatures of about 40° C. to about 50° C.; buffer concentrations ofabout 9×SSC to about 2×SSC; formamide concentrations of about 30% toabout 50%; and wash solutions of about 5×SSC to about 2×SSC. Examples ofhigh stringency conditions include: incubation temperatures of about 55°C. to about 68° C.; buffer concentrations of about 1×SSC to about0.1×SSC; formamide concentrations of about 55% to about 75%; and washsolutions of about 1×SSC, 0.1×SSC, or deionized water. In general,hybridization incubation times are from 5 minutes to 24 hours, with 1,2, or more washing steps, and wash incubation times are about 1, 2, or15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It isunderstood that equivalents of SSC using other buffer systems can beemployed.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in an eukaryotic cell.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

The term “stop codon” intends a three nucleotide contiguous sequencewithin messenger RNA that signals a termination of translation.Non-limiting examples include in RNA, UAG, UAA, UGA and in DNA TAG, TAAor TGA. Unless otherwise noted, the term also includes nonsensemutations within DNA or RNA that introduce a premature stop codong,causing any resulting protein to be abnormally shortened. For example,one can remove all the nucleotides encoding for valine, and then reducein positions where they are not typically located within the wild-typesequence.

A “nonribosomal peptide” or “NRP” is a peptide that is not synthesizedby the ribosome.

An intracellular PPI is an intracellular protein-protein interaction(“PPI”).

As used herein, the term “pre-determined specificity” intends a peptidehaving a selected ability to specifically recognize and bind apre-selected target, e.g., Her-2 receptor. Non-limiting examples ofpre-selected targets include for example, any peptide, antigen,polynucleotide or antibody.

As used herein, the term “bioavailable peptide” means a peptide,polypeptide or protein that is capable of crossing the variousbiological barriers to reach target cells after its administration, andparticularly to pass through the intestinal barrier after oraladministration. Bioavailability is determined for a selected cell typeas a function of the envisaged application. Methods for determiningbioavailability are known in the art and described herein.

As used herein, the term “stable peptide” intends a peptide, polypeptideor protein that has a lifetime, once administered in vivo, which issufficient to reach target cells and to exert its biological action.These peptides have a conformation which protects them againstdegradation by cell proteases while retaining biological activity. Anindication of the stability of a peptide may be obtained using testscarried out in vitro. For example, in vitro degradation of a peptide ismeasured by contact with a variety of purified proteases, which arecommercially available, for increasing incubation periods (1 hour to 72hours, for example). Peptide degradation is then demonstrated by reversephase HPLC, comparing the profiles obtained before and after digestion.In one aspect, the stable protein is more resistant to proteases presentin human serum, e.g., more than about 20%, or alternatively, more thanabout 40%, or alternatively more than about 50%, or alternatively morethan about 60%, or alternatively than about 70%, or alternatively morethan about 75%, or alternatively more than 80% more resistant.

As used herein, the term “mRNA library” intends a plurality of at leasttwo RNA members having a promoter region followed, by the amino acidMet, followed by a number of randomized amino acids (typically 7 to 10),followed by lysine.

A “biotin analog” as used herein intends a peptide sequence conjugatedat or near the N or C terminus for the purpose of enhancing membranepermeability, cell penetration, and/or oral availability. Non-limitingexamples of biotin analogs include, dehydrobiotin, iminobiotin,biotinamine, diaminobiotin, desthiobiotin, halogenated biotin, longchain biotin, and biotin-PEG. Methods to PEGylating peptides are knownin the art and described, for example in Mero et al., BioconjugationProtocols Methods in Molecular Biology 2011, Vol. 751, Part. 1, 95-129;and Gonen-Wadmanya et al. (2011) Biomaterials 32(26):6025-6033.

As used herein, the term “predetermined specificity” intends a selectedability to specifically recognize and bind a predetermined target, e.g.,the Her-2 receptor.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disorder or sign or symptom thereof, and/or maybe therapeutic in terms of a partial or complete cure for a disorderand/or adverse effect attributable to the disorder.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

“Pharmaceutically acceptable carriers” refers to any diluents,excipients, or carriers that may be used in the compositions of theinvention. Pharmaceutically acceptable carriers include ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances, such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. They are preferably selected with respect to theintended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

“Administration” can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents are known in the art. Route ofadministration can also be determined and method of determining the mosteffective route of administration are known to those of skill in the artand will vary with the composition used for treatment, the purpose ofthe treatment, the health condition or disease stage of the subjectbeing treated, and target cell or tissue. Non-limiting examples of routeof administration include oral administration, nasal administration,injection, and topical application.

The term “effective amount” refers to a quantity sufficient to achieve adesired effect. In the context of therapeutic or prophylacticapplications, the effective amount will depend on the type and severityof the condition at issue and the characteristics of the individualsubject, such as general health, age, sex, body weight, and tolerance topharmaceutical compositions. In the context of an immunogeniccomposition, in some embodiments the effective amount is the amountsufficient to result in a protective response against a pathogen. Inother embodiments, the effective amount of an immunogenic composition isthe amount sufficient to result in antibody generation against theantigen. In some embodiments, the effective amount is the amountrequired to confer passive immunity on a subject in need thereof. Withrespect to immunogenic compositions, in some embodiments the effectiveamount will depend on the intended use, the degree of immunogenicity ofa particular antigenic compound, and the health/responsiveness of thesubject's immune system, in addition to the factors described above. Theskilled artisan will be able to determine appropriate amounts dependingon these and other factors.

In the case of an in vitro application, in some embodiments theeffective amount will depend on the size and nature of the applicationin question. It will also depend on the nature and sensitivity of the invitro target and the methods in use. The skilled artisan will be able todetermine the effective amount based on these and other considerations.The effective amount may comprise one or more administrations of acomposition depending on the embodiment.

A “peptide conjugate” refers to the association by covalent ornon-covalent bonding of one or more polypeptides and another chemical orbiological compound. In a non-limiting example, the “conjugation” of apolypeptide with a chemical compound results in improved stability orefficacy of the polypeptide for its intended purpose. In one embodiment,a peptide is conjugated to a carrier, wherein the carrier is a liposome,a micelle, or a pharmaceutically acceptable polymer.

A “gene delivery vehicle” is defined as any molecule that can carryinserted polynucleotides into a host cell. Examples of gene deliveryvehicles are liposomes, micelles biocompatible polymers, includingnatural polymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; and bacteria, or viruses, such as baculovirus, adenovirus andretrovirus, bacteriophage, cosmid, plasmid, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

A polynucleotide of this invention can be delivered to a cell or tissueusing a gene delivery vehicle. “Gene delivery,” “gene transfer,”“transducing,” and the like as used herein, are terms referring to theintroduction of an exogenous polynucleotide (sometimes referred to as a“transgene”) into a host cell, irrespective of the method used for theintroduction. Such methods include a variety of well-known techniquessuch as vector-mediated gene transfer (by, e.g., viralinfection/transfection, or various other protein-based or lipid-basedgene delivery complexes) as well as techniques facilitating the deliveryof “naked” polynucleotides (such as electroporation, “gene gun” deliveryand various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein.

A “plasmid” is an extra-chromosomal DNA molecule separate from thechromosomal DNA which is capable of replicating independently of thechromosomal DNA. In many cases, it is circular and double-stranded.Plasmids provide a mechanism for horizontal gene transfer within apopulation of microbes and typically provide a selective advantage undera given environmental state. Plasmids may carry genes that provideresistance to naturally occurring antibiotics in a competitiveenvironmental niche, or alternatively the proteins produced may act astoxins under similar circumstances.

“Plasmids” used in genetic engineering are called “plasmid vectors”.Many plasmids are commercially available for such uses. The gene to bereplicated is inserted into copies of a plasmid containing genes thatmake cells resistant to particular antibiotics and a multiple cloningsite (MCS, or polylinker), which is a short region containing severalcommonly used restriction sites allowing the easy insertion of DNAfragments at this location. Another major use of plasmids is to makelarge amounts of proteins. In this case, researchers grow bacteriacontaining a plasmid harboring the gene of interest. Just as thebacterium produces proteins to confer its antibiotic resistance, it canalso be induced to produce large amounts of proteins from the insertedgene. This is a cheap and easy way of mass-producing a gene or theprotein it then codes for.

A “yeast artificial chromosome” or “YAC” refers to a vector used toclone large DNA fragments (larger than 100 kb and up to 3000 kb). It isan artificially constructed chromosome and contains the telomeric,centromeric, and replication origin sequences needed for replication andpreservation in yeast cells. Built using an initial circular plasmid,they are linearized by using restriction enzymes, and then DNA ligasecan add a sequence or gene of interest within the linear molecule by theuse of cohesive ends. Yeast expression vectors, such as YACs, YIps(yeast integrating plasmid), and YEps (yeast episomal plasmid), areextremely useful as one can get eukaryotic protein products withposttranslational modifications as yeasts are themselves eukaryoticcells, however YACs have been found to be more unstable than BACs,producing chimeric effects.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors, alphavirus vectors and the like. Infectious tobacco mosaicvirus (TMV)-based vectors can be used to manufacturer proteins and havebeen reported to express Griffithsin in tobacco leaves (O'Keefe et al.(2009) Proc. Nat. Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors,such as Semliki Forest virus-based vectors and Sindbis virus-basedvectors, have also been developed for use in gene therapy andimmunotherapy. See, Schlesinger & Dubensky (1999) Curr. Opin.Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. Inaspects where gene transfer is mediated by a retroviral vector, a vectorconstruct refers to the polynucleotide comprising the retroviral genomeor part thereof, and a therapeutic gene.

As used herein, “retroviral mediated gene transfer” or “retroviraltransduction” carries the same meaning and refers to the process bywhich a gene or nucleic acid sequences are stably transferred into thehost cell by virtue of the virus entering the cell and integrating itsgenome into the host cell genome. The virus can enter the host cell viaits normal mechanism of infection or be modified such that it binds to adifferent host cell surface receptor or ligand to enter the cell. Asused herein, retroviral vector refers to a viral particle capable ofintroducing exogenous nucleic acid into a cell through a viral orviral-like entry mechanism.

Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof, and a transgene. Adenoviruses (Ads) are a relatively wellcharacterized, homogenous group of viruses, including over 50 serotypes.See, e.g., PCT Publication No. WO 95/27071. Ads do not requireintegration into the host cell genome. Recombinant Ad derived vectors,particularly those that reduce the potential for recombination andgeneration of wild-type virus, have also been constructed. See, PCTPublication Nos. WO 95/00655 and WO 95/11984. Wild-type AAV has highinfectivity and specificity integrating into the host cell's genome.See, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are well known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression. DNA virus, RNAvirus, modifications, liposomes are non-limiting examples of vectors.

Gene delivery vehicles also include DNA/liposome complexes, micelles andtargeted viral protein-DNA complexes. Liposomes that also comprise atargeting antibody or fragment thereof can be used in the methods ofthis invention. In addition to the delivery of polynucleotides to a cellor cell population, direct introduction of the proteins described hereinto the cell or cell population can be done by the non-limiting techniqueof protein transfection, alternatively culturing conditions that canenhance the expression and/or promote the activity of the proteins ofthis invention are other non-limiting techniques.

As used herein, the terms “antibody,” “antibodies” and “immunoglobulin”includes whole antibodies and any antigen binding fragment or a singlechain thereof. Thus the term “antibody” includes any protein or peptidecontaining molecule that comprises at least a portion of animmunoglobulin molecule. The terms “antibody,” “antibodies” and“immunoglobulin” also include immunoglobulins of any isotype, fragmentsof antibodies which retain specific binding to antigen, including, butnot limited to, Fab, Fab′, F(ab)₂, Fv, scFv, dsFv, Fd fragments, dAb,VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies,tetrabodies and kappa bodies; multispecific antibody fragments formedfrom antibody fragments and one or more isolated. Examples of suchinclude, but are not limited to a complementarity determining region(CDR) of a heavy or light chain or a ligand binding portion thereof, aheavy chain or light chain variable region, a heavy chain or light chainconstant region, a framework (FR) region, or any portion thereof, atleast one portion of a binding protein, chimeric antibodies, humanizedantibodies, single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Thevariable regions of the heavy and light chains of the immunoglobulinmolecule contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies (Abs) may mediate the binding of theimmunoglobulin to host tissues.

As used herein, the term “label” intends a directly or indirectlydetectable compound or composition that is conjugated directly orindirectly to the composition to be detected, e.g., N-terminal histidinetags (N-His), magnetically active isotopes, e.g., ¹¹⁵Sn, ¹¹⁷Sn and¹¹⁹Sn, a non-radioactive isotopes such as ¹³C and ¹⁵N, polynucleotide orprotein such as an antibody so as to generate a “labeled” composition.The term also includes sequences conjugated to the polynucleotide thatwill provide a signal upon expression of the inserted sequences, such asgreen fluorescent protein (GFP) and the like. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable. The labelscan be suitable for small scale detection or more suitable forhigh-throughput screening. As such, suitable labels include, but are notlimited to magnetically active isotopes, non-radioactive isotopes,radioisotopes, fluorochromes, chemiluminescent compounds, dyes, andproteins, including enzymes. The label may be simply detected or it maybe quantified. A response that is simply detected generally comprises aresponse whose existence merely is confirmed, whereas a response that isquantified generally comprises a response having a quantifiable (e.g.,numerically reportable) value such as an intensity, polarization, and/orother property. In luminescence or fluorescence assays, the detectableresponse may be generated directly using a luminophore or fluorophoreassociated with an assay component actually involved in binding, orindirectly using a luminophore or fluorophore associated with another(e.g., reporter or indicator) component. Examples of luminescent labelsthat produce signals include, but are not limited to bioluminescence andchemiluminescence. Detectable luminescence response generally comprisesa change in, or an occurrence of, a luminescence signal. Suitablemethods and luminophores for luminescently labeling assay components areknown in the art and described for example in Haugland, Richard P.(1996) Handbook of Fluorescent Probes and Research Chemicals (6^(th)ed.). Examples of luminescent probes include, but are not limited to,aequorin and luciferases.

As used herein, the term “immunoconjugate” comprises an antibody or anantibody derivative associated with or linked to a second agent, such asa cytotoxic agent, a detectable agent, a radioactive agent, a targetingagent, a human antibody, a humanized antibody, a chimeric antibody, asynthetic antibody, a semisynthetic antibody, or a multispecificantibody.

Examples of suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, and Texas Red. Other suitable optical dyes aredescribed in the Haugland, Richard P. (1996) Handbook of FluorescentProbes and Research Chemicals (6^(th) ed.).

In another aspect, the fluorescent label is functionalized to facilitatecovalent attachment to a cellular component present in or on the surfaceof the cell or tissue such as a cell surface marker. Suitable functionalgroups, including, but not are limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to attach the fluorescent label to asecond molecule. The choice of the functional group of the fluorescentlabel will depend on the site of attachment to either a linker, theagent, the marker, or the second labeling agent.

“Eukaryotic cells” comprise all of the life kingdoms except monera. Theycan be easily distinguished through a membrane-bound nucleus. Animals,plants, fungi, and protists are eukaryotes or organisms whose cells areorganized into complex structures by internal membranes and acytoskeleton. The most characteristic membrane-bound structure is thenucleus. Unless specifically recited, the term “host” includes aeukaryotic host, including, for example, yeast, higher plant, insect andmammalian cells. Non-limiting examples of eukaryotic cells or hostsinclude simian, bovine, porcine, murine, rat, avian, reptilian andhuman.

“Prokaryotic cells” that usually lack a nucleus or any othermembrane-bound organelles and are divided into two domains, bacteria andarchaea. In addition to chromosomal DNA, these cells can also containgenetic information in a circular loop called an episome. Bacterialcells are very small, roughly the size of an animal mitochondrion (about1-2 μm in diameter and 10 μm long). Prokaryotic cells feature threemajor shapes: rod shaped, spherical, and spiral. Instead of goingthrough elaborate replication processes like eukaryotes, bacterial cellsdivide by binary fission. Examples include but are not limited toBacillus bacteria, E. coli bacterium, and Salmonella bacterium.

A “native” or “natural” antigen is a polypeptide, protein or a fragmentwhich contains an epitope, which has been isolated from a naturalbiological source, and which can specifically bind to an antigenreceptor, in particular a T cell antigen receptor (TCR), in a subject.

The terms “antigen” and “antigenic” refer to molecules with the capacityto be recognized by an antibody or otherwise act as a member of anantibody-ligand pair. “Specific binding” refers to the interaction of anantigen with the variable regions of immunoglobulin heavy and lightchains. Antibody-antigen binding may occur in vivo or in vitro. Theskilled artisan will understand that macromolecules, including proteins,nucleic acids, fatty acids, lipids, lipopolysaccharides andpolysaccharides have the potential to act as an antigen. The skilledartisan will further understand that nucleic acids encoding a proteinwith the potential to act as an antibody ligand necessarily encode anantigen. The artisan will further understand that antigens are notlimited to full-length molecules, but can also include partialmolecules. The term “antigenic” is an adjectival reference to moleculeshaving the properties of an antigen. The term encompasses substanceswhich are immunogenic, i.e., immunogens, as well as substances whichinduce immunological unresponsiveness, or anergy, i.e., anergens.

An “altered antigen” is one having a primary sequence that is differentfrom that of the corresponding wild-type antigen. Altered antigens canbe made by synthetic or recombinant methods and include, but are notlimited to, antigenic peptides that are differentially modified duringor after translation, e.g., by phosphorylation, glycosylation,cross-linking, acylation, proteolytic cleavage, linkage to an antibodymolecule, membrane molecule or other ligand. (Ferguson et al. (1988)Ann. Rev. Biochem. 57:285-320). A synthetic or altered antigen of theinvention is intended to bind to the same TCR as the natural epitope.

A “self-antigen” also referred to herein as a native or wild-typeantigen is an antigenic peptide that induces little or no immuneresponse in the subject due to self-tolerance to the antigen. An exampleof a self-antigen is the melanoma specific antigen gp100.

“SUPR” is an acronym for “Scanning Unnatural Protease Resistant”peptide, examples of such are shown in FIG. 2. A specific example is thepeptide MFYAYEYAQWSK, wherein A is N-methyl alanine (SEQ ID NO: 7).

The term “oral uptake” is synonymous with oral bioavailability, which isthe percent of a compound that gains access to the bloodstream afteroral consumption.

MODES FOR CARRYING OUT THE DISCLOSURE

Methods, mRNA, Libraries, Polypeptides and Proteins

In one aspect, this invention provides a method for selecting one ormore RNA members from an mRNA library, wherein the one or more RNAmembers encode one or more stable and bioavailable peptide(s). Themethod comprises, or alternatively consists essentially of, or yetfurther consists of, selecting from an mRNA library containing sequencesencoding peptides, wherein one or more members of the mRNA librarycontain one or more unnatural amino acids and/or stop codons, andwherein the peptides encoded by the one or more members are selected forpre-determined specificity and stability, thereby selecting for one ormore RNA that encode one or more stable and bioavailable peptide(s).Methods for selecting the one or members are described herein.Non-limiting examples include screening for an in vitro or in vivofunction, such as protease resistance and/or the ability to bind to apre-selected target such as the Her-2 receptor, the ability to disrupt aprotein-protein interaction, and/or the ability to withstand enzematicmodification.

This disclosure also provides a method for preparing one or more RNAthat encode one or more stable and bioavailable peptide(s), comprising,or alternatively consisting essentially of, or yet further consistingof, mutating a library of peptides selected for a pre-determinedspecificity to incorporate amino acids that impart stability and thenincorporating one or more stop codons. In another aspect, the methodfurther comprises, or alternatively consists essentially of, or yetconsists of, reverse translating the library of peptides to a RNAlibrary; cyclizing one or more of the individual sequences of the RNAlibrary; and selecting individual sequences of the mRNA library forprotease resistance, thereby selecting for one or more RNA that encodeone or more stable and bioavailable peptide(s). An example of apre-determined specificity is the Her-2 antigen. Another exampleexamined is GIBP binding to Gαi1. Any protein and binding partner couldwork. For example, a protein with an antibody, endogenous bindingpartner, peptide found by selection process such as ribosome, phage, ormRNA display are also within the scope of this invention. Amino acidsthat impart stability are known in the art and include withoutlimitation, N-methyl amino acids. Examples of N-methyl amino acidinclude, but are not limited to N-methyl norvaline or N-methyl alanineor alternatively any modification to an amino acid that confersstabilization, e.g., proline, D-amino acids, Beta amino acids, peptoids,and 2-aminoisobutyric acid (Aib) or any amino acid not encoded forribosomally. In one aspect, the one or more mRNAs are isolated from thelibrary.

In one aspect, the method further comprises, or alternatively consistsessentially of, or yet further consists of translating the one or moreselected RNAs to a peptide.

Using the disclosed methods, stable peptides were generated. Thus, inone aspect this disclosure provides a non-naturally occurring peptidecomprising, or alternatively consisting essentially of, or yet furtherconsisting of an amino acid sequence of the group:

a) MAVYVHYHK, wherein Position 1 is Met or (norvaline, alanine,norleucine); Position 2 is Ala or (V, M, S, T, H, K, R, Q, N, L, V, I)Position 3 is N-Methyl Norvaline or (S, T, Q, N, H, P, I, V, L, Y, F,P); Position 4 is Tyr or (V, Y, F, Q, N, S, T, H); Position 5 isN-Methyl Norvaline or (Y, F, S, T, E, D, M, A, P); Position 6 is His or(V, Y, F, Q, N); Position 7 is His or (V, F, Y, V, I, L); Position 8 isHis or any amino acid and Position 9 is Lys or (lysine derivatives e.g.Orn) (SEQ ID NO: 1);

b) MFVQVYYHK, wherein Position 1 is Met or (norvaline, norleucine,alanine); Position 2 is Phe or any amino acid; Position 3 is N-methylnorvaline or (Q, N, S, T, H, Y, F, P); Position 4 is Gln or (Y, F, V, P,S, T); Position 5 is N-methyl norvaline or (Y, F, S, T, D, E, A, M);Position 6 is Tyr or (F, H); Position 7 is Tyr or (F, L, I, V, S, T, V);Position 8 is His or (T, S); Position 9 is Lys or (lysine derivativese.g. Orn) (SEQ ID NO: 2);

c) MLHYVYVRK, wherein Position 1 is Met or (norvaline, norleucine,alanine); Position 2 is Leu or (I, V); Position 3 is His or (Y, F);Position 4 is Tyr or (F); Position 5 is N-methyl norvaline or (S, T, D,E, A, M, P); Position 6 is Tyr or (H, Q, N, L, I, V, V); Position 7 isN-methyl norvaline or (F, Y, L, I, V, H, P); Position 8 is Arg or anyamino acid; Position 9 is Lys or (lysine derivatives e.g. Orn) (SEQ IDNO: 3);

d) MVCVVLYDDK, wherein Position 1 is Met or (norvaline, norleucine,alanine); Position 2 is Val or (I, L); Position 3 is Cys, Position 4 isN-methyl norvaline or (Y, F, P, D, E, M); Position 5 is N-methylnorvaline or (Y, F, D, E, W, C, G, P); Position 6 is Leu or (Y, F, V, V,I, P, C); Position 7 is Tyr or (V, E, D); Position 8 is Asp or (S, T, E,Y, F, A, P, V); Position 9 is Asp or (E, G, L, I, V); Position 10 is Lysor (lysine derivatives e.g. Orn) (SEQ ID NO: 4);

e) MEVYEYVSLK, wherein Position 1 is Met or (norvaline, norleucine,alanine); Position 2 is Glu or any amino acid; Position 3 is N-methylnorvaline or (P, D, E, F, Y, S, T, Q, N); Position 4 is Tyr or (D, E, F,V, P); Position 5 is Glu or (D, Y, F, P, V); Position 6 is Tyr or (F, L,V, I, P, V); Position 7 is N-methyl norvaline or (F, L, V, I, P, V);Position 8 is Ser or any other amino acid; Position 9 is Leu or anyother amino acid; Position 10 is Lys or (lysine derivatives e.g. Orn)(SEQ ID NO: 5);

f) MNEYVLYVLK, wherein Position 1 is Met or (norvaline, norleucine,alanine); Position 2 is Asn or any amino acid; Position 3 is Glu or (D,I, V, L, F, Y, P, V); Position 4 is Tyr or (D, E, P, V); Position 5 isN-methyl norvaline or (D, E, F, Y, G, C, P); Position 6 is Leu or (Y, F,P, V); Position 7 is Tyr or (F, V, I, L); Position 8 is N-methylnorvaline or (S, T, Y, F, E, D, A, P); Position 9 is Leu or (K, R, I, L,V, D, E, G, S, T); Position 10 is Lys or (lysine derivatives e.g. Orn),and for each of the above, V, is as disclosed above or alternatively, anamino acid that confers stability such as an N-methyl amino acid (SEQ IDNO: 6).

The peptides can be further modified by conjugating the peptide to abiotin molecule or biotin analog through a disulfide linkage orpalmitoleic acid. Non-limiting examples of such include a reduciblebiotin molecule; a biotin on the side chain of lysine of the peptide; abiotin linked to the peptide through an amide linkage or an esterlinkage. Methods to conjugate the peptide to biotin molecules and/orbiotin analogs to the peptides are known in the art and are disclosedherein. Examples of such include without limitation, PEGylation,biotinylation or lipidation.

Additional specific examples of peptide conjugates comprise insulin orthe amino acid sequence MFYAYEYAQWSKK-mod, wherein A is N-methyl alanineand K(mod) is lysine with a modified side chain including biotin, abiotin analog or palmitoleic acid (SEQ ID NO: 8).

This disclosure also provides conjugates, comprising a linear orcyclized peptide linked at the N- and/or C-terminal to a biotin moleculeor a biotin analog through a suitable linkage, e.g., a disulfidelinkage. In one aspect the biotin analog is a reducible biotin molecule.In an another aspect, the peptide conjugate, comprises a peptide linkedto biotin on the side chain of lysine, or alternatively, a peptidelinked to biotin through an amide linkage, or alternatively a peptidelinked to biotin through an ester linkage. These peptide conjugates aresuitable for oral delivery and therefore, formulations suitable for oraldelivery also are provided herein.

Although not intending to be limited to the size of the peptide of theconjugate, some alternative embodiments include conjugates wherein thepeptide comprises between 4 and 50 amino acids of insulin, oralternatively between 4 and 30 amino acids, or yet further between 4 and100 amino acids. Examples of peptides include the amino acid sequencesidentified as 1 to 10 in Table 2.

Non-limiting examples of biotin analogs include, dehydrobiotin,iminobiotin, biotinamine, diaminobiotin, desthiobiotin, halogenatedbiotin, long chain biotin, and biotin-PEG. Methods to PEGylatingpeptides are known in the art and described, for example in Mero et al.,Bioconjugation Protocols Methods in Molecular Biology 2011, Vol. 751,Part. 1, 95-129; and Gonen-Wadmanya et al. (2011) Biomaterials32(26):6025-6033.

Alternatively, the peptide conjugates contain an unsaturated fatty acidin place of the biotin or biotin analog. An example of such ispalmitoleic acid.

The peptide conjugates of this disclosure contain a peptide having aN-methyl amino acid (e.g., N-methyl alanine or N-methyl norvaline)modified peptide or a modified amino acid whose modification confersstability on the peptide. Examples of such are described herein.

In a further aspect, the peptide conjugates described herein comprisepeptides wherein the amino acid sequence is cyclized from lysine to theN-terminus.

In another aspect the peptides and/or conjugates can be combined with acarrier, such as a pharmaceutically acceptable carrier, for diagnosticand/or therapeutic use.

In another aspect, the mRNA or the peptide is isolated. They can befurther screened for an in vivo therapeutic utility, such as the abilityto inhibit the growth or kill a cancer cell or a pre-cancerous cell.Methods to further screen the peptides for in vivo utility are disclosedherein.

Also disclosed is an isolated RNA library; an isolated peptide library;an isolated peptide; each prepared by the disclosed methods. These canbe combined with a carrier, such as a pharmaceutically acceptablecarrier, as disclosed herein.

For reproduction and expression of a polynucleotide, a peptide, aproteins and a polypeptides are obtainable by a number of processesknown to those of skill in the art, which include purification, chemicalsynthesis and recombinant methods. Polypeptides can be isolated frompreparations such as host cell systems by methods such asimmunoprecipitation with antibody, and standard techniques such as gelfiltration, ion-exchange, reversed-phase, and affinity chromatography.For such methodology, see for example Deutscher et al. (1999) Guide ToProtein Purification: Methods In Enzymology (Vol. 182, Academic Press).Accordingly, this disclosure also provides the processes for obtainingthese polypeptides as well as the products obtainable and obtained bythese processes.

The polypeptides also can be obtained by chemical synthesis using acommercially available automated peptide synthesizer such as thosemanufactured by Perkin/Elmer/Applied Biosystems, Inc., Model 430A or431A, Foster City, Calif., USA. The synthesized polypeptide can beprecipitated and further purified, for example by high performanceliquid chromatography (HPLC). Accordingly, this disclosure also providesa process for chemically synthesizing the proteins of this disclosure byproviding the sequence of the protein and reagents, such as amino acidsand enzymes and linking together the amino acids in the properorientation and linear sequence.

Alternatively, the proteins and polypeptides can be obtained bywell-known recombinant methods as described, for example, in Sambrook etal. (1989) supra, using a host cell and vector systems described herein.Polypeptides can be isolated from preparations such as host cell systemsby methods such as immunoprecipitation with antibody, and standardtechniques such as gel filtration, ion-exchange, reversed-phase, andaffinity chromatography. For such methodology, see for example Deutscheret al. (1999) Guide To Protein Purification: Methods In Enzymology (Vol.182, Academic Press). Accordingly, this disclosure also provides theprocesses for obtaining these polypeptides as well as the productsobtainable and obtained by these processes.

Also provided by this application are the peptides described hereinconjugated to a detectable agent for use in therapeutic or diagnosticmethods. For example, detectably labeled peptides can be bound to acolumn and used for the detection and purification of antibodies. Theyalso are useful as immunogens for the production of antibodies. Thepeptides of this disclosure are useful in an in vitro assay system toscreen for agents or drugs, which modulate cellular processes. Forexample, detectably labeled peptides can be bound to a column and usedfor the detection and purification of antibodies.

It is well know to those skilled in the art that modifications can bemade to the peptides of the disclosure to provide them with alteredproperties. As used herein the term “amino acid” refers to eithernatural and/or unnatural or synthetic amino acids, including glycine andboth the D or L optical isomers, and amino acid analogs andpeptidomimetics. A peptide of three or more amino acids is commonlycalled an oligopeptide if the peptide chain is short. If the peptidechain is long, the peptide is commonly called a polypeptide or aprotein.

Peptides of the disclosure can be modified to include unnatural aminoacids. Thus, the peptides may comprise D-amino acids, a combination ofD- and L-amino acids, and various “designer” amino acids (e.g.,.beta.-methyl amino acids, C-.alpha.-methyl amino acids, andN-.alpha.-methyl amino acids, etc.) to convey special properties topeptides. Additionally, by assigning specific amino acids at specificcoupling steps, peptides with .alpha.-helices .beta. turns, .beta.sheets, .gamma.-turns, and cyclic peptides can be generated. Generally,it is believed that .alpha.-helical secondary structure or randomsecondary structure is preferred.

The peptides of this disclosure also can be combined with various solidphase carriers, such as an implant, a stent, a paste, a gel, a dentalimplant, or a medical implant or liquid phase carriers, such as beads,sterile or aqueous solutions, pharmaceutically acceptable carriers,pharmaceutically acceptable polymers, liposomes, micelles, suspensionsand emulsions. Examples of non-aqueous solvents include propyl ethyleneglycol, polyethylene glycol and vegetable oils.

The peptides of this invention can further comprise a carrier such as apharmaceutically acceptable carrier. In one aspect, the carrier is onethat is suitable for oral administration.

In one aspect, the peptides are useful for treating cancer orseparately, regulating blood sugar or treating diabetes, prediabetes oran associated condition or disorder in a subject in need of suchtreatment, comprising administering to the subject an effective amountof a suitable peptide, polypeptide, polynucleotide, conjugate orcomposition of this disclosure. In one aspect, the subject is a humanpatient.

Also provided are method for determining if a candidate agent is apotential therapeutic suitable for oral administration, the methodcomprising administering the candidate agent to a subject and assayingfor bioavailability, and comparing the bioavailability of the candidateagent with the bioavailability of the peptide conjugate of thisdisclosure, wherein the candidate agent is a potential therapeutic ifthe bioavailability of the agent is at least substantially theequivalent of that of the peptide conjugate of this disclosure.

Further provided are kits for determining if a candidate agent is apotential therapeutic suitable for oral administration, comprising, oralternatively consisting essentially of, or yet further consisting of,the peptide conjugate as described herein and instructions for use.

This disclosure also provides a pharmaceutical composition comprising oralternatively consisting essentially of, or yet further consisting of,any of a peptide, analog, mutein, or fragment of this disclosure, aloneor in combination with each other or other agents, and an acceptablecarrier or solid support. These compositions are useful for variousdiagnostic and therapeutic methods as described herein.

Polynucleotides

This disclosure also provides isolated or recombinant polynucleotidesencoding one or more of the above-identified peptides and theirrespective complementary strands. Vectors comprising the isolated orrecombinant polynucleotides are further provided examples of which areknown in the art and briefly described herein. In one aspect where morethan one isolated or recombinant polynucleotide is to be expressed as asingle unit, the isolated or recombinant polynucleotides can becontained within a polycistronic vector. The polynucleotides can be DNA,RNA, mRNA or interfering RNA, such as siRNA, miRNA or dsRNA.

The disclosure further provides the isolated or recombinantpolynucleotide operatively linked to a promoter of RNA transcription, aswell as other regulatory sequences for replication and/or transient orstable expression of the DNA or RNA. As used herein, the term“operatively linked” means positioned in such a manner that the promoterwill direct transcription of RNA off the DNA molecule. Examples of suchpromoters are SP6, T4 and T7. In certain embodiments, cell-specificpromoters are used for cell-specific expression of the insertedpolynucleotide. Vectors which contain a promoter or a promoter/enhancer,with termination codons and selectable marker sequences, as well as acloning site into which an inserted piece of DNA can be operativelylinked to that promoter are known in the art and commercially available.For general methodology and cloning strategies, see Gene ExpressionTechnology (Goeddel ed., Academic Press, Inc. (1991)) and referencescited therein and Vectors: Essential Data Series (Gacesa and Ramji,eds., John Wiley & Sons, N.Y. (1994)) which contains maps, functionalproperties, commercial suppliers and a reference to GenEMBL accessionnumbers for various suitable vectors.

In one embodiment, polynucleotides derived from the polynucleotides ofthe disclosure encode polypeptides or proteins having diagnostic andtherapeutic utilities as described herein as well as probes to identifytranscripts of the protein that may or may not be present.

Expression vectors containing these nucleic acids are useful to obtainhost vector systems to produce proteins and polypeptides. It is impliedthat these expression vectors must be replicable in the host organismseither as episomes or as an integral part of the chromosomal DNA.Non-limiting examples of suitable expression vectors include plasmids,yeast vectors, viral vectors and liposomes. Adenoviral vectors areparticularly useful for introducing genes into tissues in vivo becauseof their high levels of expression and efficient transformation of cellsboth in vitro and in vivo. When a nucleic acid is inserted into asuitable host cell, e.g., a prokaryotic or a eukaryotic cell and thehost cell replicates, the protein can be recombinantly produced.Suitable host cells will depend on the vector and can include mammaliancells, animal cells, human cells, simian cells, insect cells, yeastcells, and bacterial cells constructed using known methods. SeeSambrook, et al. (1989) supra. In addition to the use of viral vectorfor insertion of exogenous nucleic acid into cells, the nucleic acid canbe inserted into the host cell by methods known in the art such astransformation for bacterial cells; transfection using calcium phosphateprecipitation for mammalian cells; or DEAE-dextran; electroporation; ormicroinjection. See, Sambrook et al. (1989) supra, for methodology.Thus, this disclosure also provides a host cell, e.g. a mammalian cell,an animal cell (rat or mouse), a human cell, or a prokaryotic cell suchas a bacterial cell, containing a polynucleotide encoding a protein orpolypeptide or antibody.

When the vectors are used for gene therapy in vivo or ex vivo, apharmaceutically acceptable vector is preferred, such as areplication-incompetent retroviral or adenoviral vector.Pharmaceutically acceptable vectors containing the nucleic acids of thisdisclosure can be further modified for transient or stable expression ofthe inserted polynucleotide. As used herein, the term “pharmaceuticallyacceptable vector” includes, but is not limited to, a vector or deliveryvehicle having the ability to selectively target and introduce thenucleic acid into dividing cells. An example of such a vector is a“replication-incompetent” vector defined by its inability to produceviral proteins, precluding spread of the vector in the infected hostcell. An example of a replication-incompetent retroviral vector is LNL6(Miller et al. (1989) BioTechniques 7:980-990). The methodology of usingreplication-incompetent retroviruses for retroviral-mediated genetransfer of gene markers has been established. (Bordignon (1989) PNASUSA 86:8912-8952; Culver (1991) PNAS USA 88:3155; and Rill (1991) Blood79(10):2694-2700).

This disclosure also provides genetically modified cells that containand/or express the polynucleotides or polypeptides of this disclosure.The genetically modified cells can be produced by insertion of upstreamregulatory sequences such as promoters or gene activators (see, U.S.Pat. No. 5,733,761), or by insertion of the peptides of this disclosure.

The polynucleotides also can be conjugated to a detectable marker, e.g.,an enzymatic label or a radioisotope for detection of nucleic acidand/or expression of the gene in a cell. A wide variety of appropriatedetectable markers are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In one aspect, one willlikely desire to employ a fluorescent label or an enzyme tag, such asurease, alkaline phosphatase or peroxidase, instead of radioactive orother environmentally undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates can be employed to provide a meansvisible to the human eye or spectrophotometrically, to identify specifichybridization with complementary nucleic acid-containing samples. Thus,this disclosure further provides a method for detecting asingle-stranded polynucleotide or its complement, by contacting targetsingle-stranded polynucleotide with a labeled, single-strandedpolynucleotide (a probe) which is a portion of the polynucleotide ofthis disclosure under conditions permitting hybridization (preferablymoderately stringent hybridization conditions) of complementarysingle-stranded polynucleotides, or more preferably, under highlystringent hybridization conditions. Hybridized polynucleotide pairs areseparated from un-hybridized, single-stranded polynucleotides. Thehybridized polynucleotide pairs are detected using methods known tothose of skill in the art and set forth, for example, in Sambrook et al.(1989) supra.

The polynucleotide embodied in this disclosure can be obtained usingchemical synthesis, recombinant cloning methods, PCR, or any combinationthereof. Methods of chemical polynucleotide synthesis are known in theart and need not be described in detail herein. One of skill in the artcan use the sequence data provided herein to obtain a desiredpolynucleotide by employing a DNA synthesizer or ordering from acommercial service.

One method to amplify polynucleotides is PCR and kits for PCRamplification are commercially available. After amplification, theresulting DNA fragments can be detected by any appropriate method knownin the art, e.g., by agarose gel electrophoresis followed byvisualization with ethidium bromide staining and ultravioletillumination.

The polynucleotides of this disclosure can be isolated or replicatedusing PCR. The PCR technology is the subject matter of U.S. Pat. Nos.4,683,195; 4,800,159; 4,754,065; and 4,683,202 and described in PCR: ThePolymerase Chain Reaction (Mullis et al. eds., Birkhauser Press, Boston(1994)) or MacPherson et al. (1991) and (1995) supra, and referencescited therein. Alternatively, one of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to replicatethe DNA. Accordingly, this disclosure also provides a process forobtaining the polynucleotides of this disclosure by providing the linearsequence of the polynucleotide, nucleotides, appropriate primermolecules, chemicals such as enzymes and instructions for theirreplication and chemically replicating or linking the nucleotides in theproper orientation to obtain the polynucleotides. In a separateembodiment, these polynucleotides are further isolated. Still further,one of skill in the art can insert the polynucleotide into a suitablereplication vector and insert the vector into a suitable host cell(prokaryotic or eukaryotic) for replication and amplification. The DNAso amplified can be isolated from the cell by methods known to those ofskill in the art. A process for obtaining polynucleotides by this methodis further provided herein as well as the polynucleotides so obtained.

Alternatively, RNA can be obtained by first inserting a DNApolynucleotide into a suitable host cell. The DNA can be delivered byany appropriate method, e.g., by the use of an appropriate gene deliveryvehicle (e.g., liposome, plasmid or vector) or by electroporation. Whenthe cell replicates and the DNA is transcribed into RNA; the RNA canthen be isolated using methods known to those of skill in the art, forexample, as set forth in Sambrook et al. (1989) supra. For instance,mRNA can be isolated using various lytic enzymes or chemical solutionsaccording to the procedures set forth in Sambrook et al. (1989) supra,or extracted by nucleic-acid-binding resins following the accompanyinginstructions provided by manufactures.

Polynucleotides exhibiting sequence complementarity or homology to apolynucleotide of this disclosure are useful as hybridization probes oras an equivalent of the specific polynucleotides identified herein.Since the full coding sequence of the transcript is known, any portionof this sequence or homologous sequences, can be used in the methods ofthis disclosure.

It is known in the art that a “perfectly matched” probe is not neededfor a specific hybridization. Minor changes in probe sequence achievedby substitution, deletion or insertion of a small number of bases do notaffect the hybridization specificity. In general, as much as 20%base-pair mismatch (when optimally aligned) can be tolerated.Preferably, a probe useful for detecting the aforementioned mRNA is atleast about 80% identical to the homologous region. More preferably, theprobe is 85% identical to the corresponding gene sequence afteralignment of the homologous region; even more preferably, it exhibits90% identity.

These probes can be used in radioassays (e.g. Southern and Northern blotanalysis) to detect, prognose, diagnose or monitor various cells ortissues containing these cells. The probes also can be attached to asolid support or an array such as a chip for use in high throughputscreening assays for the detection of expression of the genecorresponding a polynucleotide of this disclosure. Accordingly, thisdisclosure also provides a probe comprising or corresponding to apolynucleotide of this disclosure, or its equivalent, or its complement,or a fragment thereof, attached to a solid support for use in highthroughput screens.

The total size of fragment, as well as the size of the complementarystretches, will depend on the intended use or application of theparticular nucleic acid segment. Smaller fragments will generally finduse in hybridization embodiments, wherein the length of thecomplementary region may be varied, such as between at least 5 to 10 toabout 100 nucleotides, or even full length according to thecomplementary sequences one wishes to detect.

Nucleotide probes having complementary sequences over stretches greaterthan 5 to 10 nucleotides in length are generally preferred, so as toincrease stability and selectivity of the hybrid, and thereby improvingthe specificity of particular hybrid molecules obtained. Morepreferably, one can design polynucleotides having gene-complementarystretches of 10 or more or more than 50 nucleotides in length, or evenlonger where desired. Such fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, byapplication of nucleic acid reproduction technology, such as the PCRtechnology with two priming oligonucleotides as described in U.S. Pat.No. 4,603,102 or by introducing selected sequences into recombinantvectors for recombinant production. In one aspect, a probe is about50-75 or more alternatively, 50-100, nucleotides in length.

The polynucleotides of the present disclosure can serve as primers forthe detection of genes or gene transcripts that are expressed in cellsdescribed herein. In this context, amplification means any methodemploying a primer-dependent polymerase capable of replicating a targetsequence with reasonable fidelity. Amplification may be carried out bynatural or recombinant DNA-polymerases such as T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Forillustration purposes only, a primer is the same length as thatidentified for probes.

Methods for administering an effective amount of a gene delivery vectoror vehicle to a cell have been developed and are known to those skilledin the art and described herein. Methods for detecting gene expressionin a cell are known in the art and include techniques such as inhybridization to DNA microarrays, in situ hybridization, PCR, RNaseprotection assays and Northern blot analysis. Such methods are useful todetect and quantify expression of the gene in a cell. Alternativelyexpression of the encoded polypeptide can be detected by variousmethods. In particular it is useful to prepare polyclonal or monoclonalantibodies that are specifically reactive with the target polypeptide.Such antibodies are useful for visualizing cells that express thepolypeptide using techniques such as immunohistology, ELISA, and Westernblotting. These techniques can be used to determine expression level ofthe expressed polynucleotide.

Compositions

Compositions are further provided. The compositions comprise a carrierand one or more of an isolated mRNA of the disclosure, an isolatedpolypeptide of the disclosure, an isolated polynucleotide of thedisclosure, a vector of the disclosure, an isolated host cell of thedisclosure, or an antibody of the disclosure. The carriers can be one ormore of a solid support or a pharmaceutically acceptable carrier. Thecompositions can further comprise an adjuvant or other componentssuitable for administrations as vaccines. In one aspect, thecompositions are formulated with one or more pharmaceutically acceptableexcipients, diluents, carriers and/or adjuvants. In addition,embodiments of the compositions of the present disclosure include one ormore of an isolated polypeptide of the disclosure, an isolatedpolynucleotide of the disclosure, a vector of the disclosure, anisolated host cell of the disclosure, or an antibody of the disclosure,formulated with one or more pharmaceutically acceptable auxiliarysubstances.

For oral preparations, any one or more of an isolated or recombinantpolypeptide as described herein, an isolated or recombinantpolynucleotide as described herein, a vector as described herein, anisolated host cell as described herein, can be used alone or inpharmaceutical formulations of the disclosure comprising, or consistingessentially of, the peptide or other agent of this disclosure incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition. The tablets, pills, capsules, troches and thelike can contain any of the following ingredients, or compounds of asimilar nature: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; alubricant such as magnesium stearate or Sterotes; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring.

Pharmaceutical formulations and unit dose forms suitable for oraladministration are particularly useful in the treatment of chronicconditions, infections, and therapies in which the patientself-administers the drug. In one aspect, the formulation is specificfor pediatric administration.

The disclosure provides pharmaceutical formulations in which the one ormore of an isolated peptide of the disclosure, an isolatedpolynucleotide of the disclosure, a vector of the disclosure, anisolated host cell of the disclosure, can be formulated intopreparations for administration in accordance with the disclosure bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives or other anticancer agents. For intravenousadministration, suitable carriers include physiological saline, orphosphate buffered saline (PBS). In all cases, a composition forparenteral administration must be sterile and should be fluid to theextent that easy syringability exists.

Aerosol formulations provided by the disclosure can be administered viainhalation and can be propellant or non-propellant based. For example,embodiments of the pharmaceutical formulations of the disclosurecomprise a peptide of the disclosure formulated into pressurizedacceptable propellants such as dichlorodifluoromethane, propane,nitrogen and the like. For administration by inhalation, the compoundscan be delivered in the form of an aerosol spray from a pressurizedcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. A non-limiting example of anon-propellant is a pump spray that is ejected from a closed containerby means of mechanical force (i.e., pushing down a piston with one'sfinger or by compression of the container, such as by a compressiveforce applied to the container wall or an elastic force exerted by thewall itself (e.g. by an elastic bladder)).

Suppositories of the disclosure can be prepared by mixing a compound ofthe disclosure with any of a variety of bases such as emulsifying basesor water-soluble bases. Embodiments of this pharmaceutical formulationof a compound of the disclosure can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration, such as syrups,elixirs, and suspensions, may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the disclosure. Similarly, unit dosage forms for injection orintravenous administration may comprise a compound of the disclosure ina composition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

Embodiments of the pharmaceutical formulations of the disclosure includethose in which one or more of an isolated polypeptide of the disclosure,an isolated polynucleotide of the disclosure, a vector of thedisclosure, an isolated host cell of the disclosure, or an antibody ofthe disclosure is formulated in an injectable composition. Injectablepharmaceutical formulations of the disclosure are prepared as liquidsolutions or suspensions; or as solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection. The preparation mayalso be emulsified or the active ingredient encapsulated in liposomevehicles in accordance with other embodiments of the pharmaceuticalformulations of the disclosure.

In an embodiment, one or more of an isolated polypeptide of thedisclosure, an isolated polynucleotide of the disclosure, a vector ofthe disclosure, an isolated host cell of the disclosure, or an antibodyof the disclosure is formulated for delivery by a continuous deliverysystem. The term “continuous delivery system” is used interchangeablyherein with “controlled delivery system” and encompasses continuous(e.g., controlled) delivery devices (e.g., pumps) in combination withcatheters, injection devices, and the like, a wide variety of which areknown in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present disclosure. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; andthe like. In general, delivery of a compound of the disclosure can beaccomplished using any of a variety of refillable, pump systems. Pumpsprovide consistent, controlled release over time. In some embodiments, acompound of the disclosure is in a liquid formulation in adrug-impermeable reservoir, and is delivered in a continuous fashion tothe individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to, a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are used in some embodimentsbecause of convenience in implantation and removal of the drug deliverydevice.

Drug release devices suitable for use in the disclosure may be based onany of a variety of modes of operation. For example, the drug releasedevice can be based upon a diffusive system, a convective system, or anerodible system (e.g., an erosion-based system). For example, the drugrelease device can be an electrochemical pump, osmotic pump, anelectroosmotic pump, a vapor pressure pump, or osmotic bursting matrix,e.g., where the drug is incorporated into a polymer and the polymerprovides for release of drug formulation concomitant with degradation ofa drug-impregnated polymeric material (e.g., a biodegradable,drug-impregnated polymeric material). In other embodiments, the drugrelease device is based upon an electrodiffusion system, an electrolyticpump, an effervescent pump, a piezoelectric pump, a hydrolytic system,etc.

Drug release devices based upon a mechanical or electromechanicalinfusion pump can also be suitable for use with the present disclosure.Examples of such devices include those described in, for example, U.S.Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and thelike. In general, a subject treatment method can be accomplished usingany of a variety of refillable, non-exchangeable pump systems. Pumps andother convective systems are generally preferred due to their generallymore consistent, controlled release over time. Osmotic pumps are used insome embodiments due to their combined advantages of more consistentcontrolled release and relatively small size (see, e.g., PCT PublicationNo. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396). Exemplaryosmotically-driven devices suitable for use in the disclosure include,but are not necessarily limited to, those described in U.S. Pat. Nos.3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631;3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440;4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318;5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; andthe like. A further exemplary device that can be adapted for the presentdisclosure is the Synchromed infusion pump (Medtronic).

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted herein,an implantation site is a site within the body of a subject at which adrug delivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

Suitable excipient vehicles for a peptide of the disclosure are, forexample, water, saline, dextrose, glycerol, ethanol, or the like, andcombinations thereof. In addition, if desired, the vehicle may containminor amounts of auxiliary substances such as wetting or emulsifyingagents or pH buffering agents. Methods of preparing such dosage formsare known, or will be apparent upon consideration of this disclosure, tothose skilled in the art. See, e.g., Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. Thecomposition or formulation to be administered will, in any event,contain a quantity of the compound adequate to achieve the desired statein the subject being treated.

Compositions of the present disclosure include those that comprise asustained-release or controlled release matrix. In addition, embodimentsof the present disclosure can be used in conjunction with othertreatments that use sustained-release formulations. As used herein, asustained-release matrix is a matrix made of materials, usuallypolymers, which are degradable by enzymatic or acid-based hydrolysis orby dissolution. After administration, the matrix is acted upon byenzymes and body fluids. A sustained-release matrix desirably is chosenfrom biocompatible materials such as liposomes, polylactides (polylacticacid), polyglycolide (polymer of glycolic acid), polylactideco-glycolide (copolymers of lactic acid and glycolic acid),polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid,collagen, chondroitin sulfate, carboxcylic acids, fatty acids,phospholipids, polysaccharides, nucleic acids, polyamino acids, aminoacids such as phenylalanine, tyrosine, isoleucine, polynucleotides,polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrativebiodegradable matrices include a polylactide matrix, a polyglycolidematrix, and a polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) matrix.

In another embodiment, the peptide (as well as combination compositions)is delivered in a controlled release system. For example, a peptide ofthe disclosure may be administered using intravenous infusion, animplantable osmotic pump, a transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump may be used (Sefton (1987)CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In anotherembodiment, polymeric materials are used. In yet another embodiment acontrolled release system is placed in proximity of the therapeutictarget, i.e., the liver, thus requiring only a fraction of the systemicdose.

In another embodiment, the compositions of the present disclosure (aswell as combination compositions separately or together) include thoseformed by impregnation of a peptide described herein into absorptivematerials, such as sutures, bandages, and gauze, or coated onto thesurface of solid phase materials, such as surgical staples, zippers andcatheters to deliver the compositions. Other delivery systems of thistype will be readily apparent to those skilled in the art in view of theinstant disclosure.

Therapeutic Methods

Further provided are methods for treating a subject in need oftreatment, comprising consisting essentially of, or yet furtherconsisting of, administering to the subject an effective amount of apeptide obtainable by the methods of this disclosure, the conjugate, ora composition of this disclosure, or a combination of any thereof.

Also provided by this disclosure are therapeutic methods comprising, oralternatively consisting essentially of, or yet further consisting of,contacting a target cell, such as a cancer cell, with an effectiveamount of a peptide obtainable by the methods of this disclosure, theconjugate, or a composition of this disclosure, or a combination of anythereof. Contacting can be in vitro or in vivo. When performed in vitro,the method is a useful pre-clinical screen. When the method is performedin vivo in an animal such as a mouse or other animal model, it is asecondary pre-clinical screen for the testing of candidate agents.

Also provided by this disclosure are methods for regulating blood sugaror for treating diabetes or an associated condition or disorder in asubject in need of such treatment, comprising, or alternativelyconsisting essentially of, or yet further consisting of, administeringto the subject an effective amount of a composition of this disclosure.In one aspect, the subject is a human patient, e.g., a patient sufferingfrom diabetes or an associated condition.

Combination Therapy

The compositions and related methods of the present disclosure may beused in combination with the administration of other therapies, e.g.,administration of a GLP-1 analog, described below. The additionaltherapeutic treatment can be added prior to, concurrent with, orsubsequent to methods or compositions used to treat diabetes and/orrelated conditions, and can be contained within the same formulation oras a separate formulation.

Screening Assays

The present disclosure provides methods for screening for equivalentagents, such as equivalent peptides to a peptide or composition of thisdisclosure, and various agents that modulate the activity of the activeagents and pharmaceutical compositions of the disclosure or the functionof a polypeptide or peptide product encoded by the polynucleotide ofthis disclosure. For the purposes of this disclosure, an “candidateagent” is intended to include, but not be limited to a biological orchemical compound such as a simple or complex organic or inorganicmolecule, a peptide, a protein (e.g. antibody), a polynucleotide (e.g.anti-sense) or a ribozyme. A vast array of compounds can be synthesized,for example polymers, such as polypeptides and polynucleotides, andsynthetic organic compounds based on various core structures, and theseare also included in the term “agent.” In addition, various naturalsources can provide compounds for screening, such as plant or animalextracts, and the like. It should be understood, although not alwaysexplicitly stated that the agent is used alone or in combination withanother agent, having the same or different biological activity as theagents identified by the inventive screen.

Kits

Kits containing the agents and instructions necessary to perform the invitro and in vivo methods as described herein also are claimed.Accordingly, the disclosure provides kits for performing these methodswhich may include peptides and/or other composition of this disclosureas well as instructions for carrying out the methods of this disclosuresuch as collecting tissue and/or performing the screen, and/or analyzingthe results, and/or administration of an effective amount of a peptideor other composition as described herein. These can be combined withother known or other candidate agents.

The following examples are intended to illustrate, and not limit, thedisclosures disclosed herein.

Experiment No. 1

This experiment describes the synthesis and screening of mRNA librariesthat encode protease resistant peptides and synthesis of peptides fororal administration.

Gαi1 Expression and Immobilization.

2 L Gαi1 was expressed with a C-terminal BirA tag in the XL-10 goldstrain of E. Coli under standard conditions. 50 mL aliquots of thegrowth were spun down at 6000 rpm at 4° C. and the pellets were washedwith ice cold PBS and stored at −80° C. Prior to each round ofselection, a pellet is lysed with 1 mL of BPER. 120 uL of NeutrAvidinagarose beads are used for immobilization. Immobilization takes place inbinding buffer with 1 mM PMSF for 1 hr at 4° C. The remaining biotinbinding sites are quenched by the addition of 1 mM biotin for 30 min.The beads are then stored in binding buffer at 4° C. for up to one week.

THG73 tRNA Preparation.

The sequence 5′-ATT ATG CTG AGT GAT ATC CAA GAT ATC ATA TCG CCA ATC ATGACC CCT GAG ATT TAG GGA ACT GGA CCC AAG CTT AGG GTC ATC CTG GAG-3′ (SEQID NO: 9) was ordered PAGE gel purified from Integrated DNATechnologies. Standard transcription was accomplished by T7 runofftranscription and purified by urea-PAGE.

NMA-tRNA.

The synthesis of N-methyl, N-nitroveratrylcarbonyl alanine cyanomethylester was carried out according to the published protocol, see forexample Millward et al. (2007), supra. The final product was purified bysilica gel chromatography in 3:1 EtOAc/hexanes. Yield 187.5 mg (24%).The synthesis of N-methyl, N-nitroveratrylcarbonyl alanine-dCA wascarried out according to Millward et al. (2007), supra. Yield % 0.5 mg(2.5%). Following ligation to THG-73 tRNA, deprotection of thenitroveratryloxycarbonyl group was effected by photolysis with a xenonlamp equipped with a 315-nm cutoff filter, and the NMAtRNA wasimmediately added to the translation reaction.

Synthesis of N-Methyl Scanning Library.

Single-stranded DNA template was ordered from Integrated DNATechnologies and the sequence was is 5′-GGG ACA ATT ACT ATT TAC AAT TACAAT GWW KWM STR GTA KKA RTW KKW GKM GKR SWM SAA ATC TGG AAG TGG AAG TGGA-3′ (SEQ ID NO: 10) using machine mixing for variable positions (W=A orT, K=G or T, M=A or C, R=G or A, S=G or C). The double stranded librarywas produced and amplified by performing 6 cycles of PCR on 1 pmole ofPAGE gel purified single-stranded library. PCR was performed understandard conditions utilizing the primers Gen-FP (5′-TAA TAC GAC TCA CTATAG GGA CAA TTA CTA TTT ACA ATT ACA-3′ (SEQ ID NO: 11)) and the reverseprimer (5′-CCA CTT CCA CTT CCA GAT TT-3′ (SEQ ID NO: 12)).

The Round 0 mRNA pool was generated by T7 runoff transcription andpurified by urea-PAGE. The purified mRNA was ligated to F30P(5=−dA21[C9]3dAdCdC-P (SEQ ID NO: 74); C9% tri-(ethylene glycol)phosphate (Glen Research), P % puromycin (Glen Research)), via anoligonucleotide splint (5′-TTT TTT TTT TTT TCC ACT TCC ACT-3′ (SEQ IDNO: 13)). Ligation was urea-PAGE purified and quantified via absorbanceat 260.

Translation and Cyclization.

50 pmol of ligation was translated in rabbit reticulocyte lysate understandard conditions. The translation reaction was supplemented with 20ug of NMAtRNA-CUA in 1 mM NaOAc (pH=4.5) and 35S-methionine. After 45min of translation at 30° C., KOAc and MgOAc were added to a finalconcentration of 600 and 50 mM, respectively, and the reactions wereincubated at room temp for 15 min. Translation mixtures were diluted 1:5in dT binding buffer (10 mg mL*1 dT cellulose, 1 M NaCl, 20 mM Tris, 1mM EDTA, 0.2% (v/v) Triton X-100, pH 8) and agitated for 45 min at 4° C.The dT cellulose was filtered and washed with dT wash buffer (300 mMNaCl, 20 mM Tris, pH 8). The conjugates were eluted with water andethanol precipitated in the presence of linear acrylamide (Ambion). Theround 0 pool was cyclized by adding 150 uL of dTpurified fusions in 50mM phosphate buffer (pH 8) to 50 μL of DSG (1 mgmL/1 in DMF). Thereaction was allowed to proceed for 1 hour, and the fusions wererepurified by ethanol precipitation.

Selection.

Following ethanol precipitation of the round 0 DSG treated fusions, thepellet was dissolved in 100 μL of dH₂O and reverse transcribed withSuperscript II under standard conditions. Following reversetranscription, the library was diluted 2 fold into 50 mM potassiumphosphate buffer (pH=8.0). Round 0 digest used 1 mg of immobilizedtrypsin, 1 mg of immobilized chymotrypsin, 1 mg of immobilizedproteinase K, and 1 mg of immobilized aminopeptidase for 20 min at roomtemp. All immobilized proteases were purchased from Sigma. Subsequentrounds used only immobilized chymotrypsin and proteinase K forproteolysis. Fusions were purified from proteases by spin filtration.

Digested fusions were put into 1.5 mLs of binding buffer (25 mMHEPES-KOH (pH % 7.5), 150 mM NaCl, 0.05% (v/v) Tween-20, 1 mM EDTA, 5 mMMgCl2, 10 μM GDP, and 0.05% (w/v) bovine serum albumin) containing 20 μLof Gαi1-NeutrAvidin agarose (preblocked with biotin). The bindingreaction was carried out at 4° C. for 1 hour. The reaction was filtered,and the resin was washed four times with 1× selection buffer. Thelibrary was eluted with 0.15% (w/v) SDS at RT, and the SDS was removedfrom the sample using SDS-Out (Pierce). Following ethanol precipitation,the library was amplified by PCR. PCR amplification of the elutedlibrary members was carried until band was observed on a 4% agarose gel(18-24 cycles).

The round 5 pool was amplified by PCR and was subcloned into the TOPO-TAvector (Invitrogen) followed by transformation into TOP10 competentcells (Invitrogen). Individual clones were sequenced (Laragen).

Peptide Synthesis.

All solvents were purchased from Sigma. C-terminally biotinylated SUPRpeptide was synthesized on Novatag resing (250 mgs, 0.12 mmoles, EMDBiosciences). Palmitoleic acid conjugated SUPR was synthesized on Wangresin preloaded with D-Lys (250 mgs, 0.1 mmoles, Anaspec). All otherpeptides were synthesized using Rink Amide Am resin (250 mgs, 0.15mmoles). Standard couplings were carried out with 5 eq. of monomer(Novabiochem) in (2 mL, 0.6 mMolar) HATU (Novabiochem) in DMF with (1.2mMolar) DIEA at room temperature for 20 min. Coupling to an N-methylamino acid followed the same procedure with the addition of HOAT (1.2mMolar) and an extended coupling time of 30 min. Fmoc deprotection wascarried out with 20% piperidine (Anaspec) at room temperature for 15min. Following, deprotection, cleavage with 95% TFA, filtration andether extraction, the crude product was purified on a Vydac C-18 reversephase column using gradient elution (0% B for 5 min, 10-50% B in 40 min.Solvent A: H₂O with 0.1% TFA), Solvent B: CH₃CN with 0.035% TFA.Lyophilized solid was reconstituted in DMSO and quantitated byabsorbance at 280 nm (ε280=9970 L mol-1 cm-1). Yield=10-25%. Cyclizationof peptide (1 mg/mL) with 20 equivalents of disuccinimidyl glutarate(DSG, Pierce) was carried out in PBS with 70% DMSO at 42° C. overnight.Peptide was purified as previously described. Yield=15-25%

Specificity Analysis.

TNT pulldown experiments were carried out as previously described inStoop and Craik (2003) Nat. Biotech 21:1063. Equal amounts of eachradiolabeled subunit were added to 10 μL of NeutrAvidin-agarosecontaining 15 pmol of prebound SUPR-Bio peptide in 1 mL of 1× selectionbuffer. Control NeutrAvidin-agarose was treated with DMSO alone. Bindingreactions proceeded for 1 h at 4° C. followed by filtration and washingwith 1× selection buffer. The matrix was analyzed by scintillationcounting, and the percent bound was determined by the matrix countsdivided by the total counts as determined by TCA precipitation.

R6A Competition/Equilibrium Binding.

The relative binding affinities of R6A and SUPR were determined byequilibrium competition experiments versus biotinylated R6A (Bio-R6A),and fits and midpoints were determined using GraphPad Prism 5.0following previously established protocols, such as Fiacco et al.(2008), supra.

Protease Resistance Experiment.

Immobilized chymotrypsin and proteinase K purchased from Sigma. 250nmoles of peptide in DMSO were added to 50 mM sodium phosphate buffer(pH=8.0) with a final DMSO concentration of 2%. Immobilized protease wasadded (60 units of chymotrypsin agarose, or 6 units of proteinase K) wasadded at room temp for chymotrypsin, and 4° C. for proteinase K.Alaquotes were taken at various time points, filtered and injected ontoa C-18 reverse phase column and separated by gradient elution (15-90% Bin 25 min. Solvent A: 0.1% TFA in water. Solvent B: CH3CN (0.05% TFA).The area under the starting material peak was quantitated using the 32KaratGold Software package (Beckman). The plotted values represent themean of two experimental values, and the error bars represent thestandard error of the mean. The graph was generated by fitting the datato a one phase exponential decay equation (GraphPad Prism 5.0).

Human Serum Digests.

Delipidated/lyophilized human serum was purchased from Thermo Scientificand reconstituted as per manufacturer's instructions. 250 nmoles ofpeptide in 50 μLs of sodium phosphate buffer (pH=8.0) with 10% DMSO wasadded to 1 mL of reconstituted serum and incubated at 37° C. 100 μLaliquots were taken at various time points and quenched in 300 μLs ofacetonitrile. Samples were spun down and decanted to remove precipitatefollowed by dilution in water to 1.5 mLs. Samples were then analyzed byHPLC as described in the protease resistance experiment.

Human Microsome Digests.

Pooled, mixed gender human microsomes containing cytochrome P450's wereobtained from Xenotech. 100 μL of microsomes were added to 500 of PBSbuffer containing 250 nmoles of peptide with 1% DMSO. 50 μL aliquotswere removed at various time points and worked up in the same manner asthe human serum digests.

In Vivo Half-Life.

Peptides synthesized as previously described with an additionalC-terminal lysine(FAM). 200 μL of peptide was administered by IVinjection into the tail vein of C57BL6 mice at a dose of 10 mg/kg. 50 μLblood samples were taken at various time points by orbital bleedingusing heparin coated capillary tubes and isoflurane for anesthesia.Samples are diluted to 600 μLin TE buffer (pH=8.0) then filtered bycentrifugation through a 3000 MWCO filter. The flowthrough was analyzedon a Shimadzu RF-5301 PC fluorometer. Time points were integrated andanalyzed using Prism 5.0 software.

Oral Biolavailability.

The following peptides were synthesized for oral administration:MFYVYEYVQWSKK(FAM) (SEQ ID NO: 14), MFYVYEYVQWSKK(FAM)DK(Palmitoleicacid) (SEQ ID NO: 15), and MITWYEFVAGTKK(FAM) (SEQ ID NO: 16), whereinFAM is a lysine derivative containing fluorescein. All peptides werecyclized from the side chain to lysine to the N-terminus as previouslydescribed. Anesthetized C57BL6 mice had peptide administered at a doseof 10 mg/kg by oral gavage. After administration, mice were removed fromisoflurane. At various time points, blood samples were taken by orbitalbleeding as previously described. Peaks were normalized to a 15 minutetime point from mice that had peptide administered by IV injection.

Quantifying Frequency of SUPR in R5 Pool.

The following DNA sequence 5′-biotin-TTT GGA CCA CTG CTA ATA CTC ATA CTAGTA AAA CAT (SEQ ID NO: 17) was the anticoding strand for FYVYEYVQWS(SEQ ID NO: 18) was ordered as a biotinylated oligo from Integrated DNATechnologies. Approximately 35 nmoles of DNA was immobilized toapproximately 265 μL of NeutrAvidin agarose beads (Pierce).Transcription from round 5 was incubated with beads containing eitherSUPR DNA or beads without DNA. As a positive control, transcription forSUPR peptide was incubated with beads containing the complementary DNA.The library and the beads were rotated in DT Buffer (50 mM HEPES-KOHpH=7.5, 1 M NaCl, 1 mM EDTA, 0.05% Tween) for 1 hour at 4° C. Absorbanceat 260 was taken before and after incubation with immobilized DNA.

Discussion

This disclosure provides methods to isolate and produce peptides thatare readily translated, and thus amenable for use in mRNA display. Thepeptides provided by these methods also exhibit a high level ofgeneralized protease resistance. In one aspect, chymotrypsin and trypsinwere incorporated because they are proteases that are abundant in theintestines. In another embodiment, aminopeptidase was chosen since itsprevalence in human serum. In a yet further embodiment, proteinase K wasincorporated for two reasons (1) as a surrogate for all proteasesgenerally due to its broad specificity and (2) because N-methylinsertion provided a high level of proteinase K resistance as reportedby Frankel et al. (2003) supra.

In one aspect, the method comprises scanning library based on cyclicGIBP (cycGIBP) although larger and smaller peptides can be used in themethods of this disclosure in which each position was systematicallycombined with either the wild type or UAG or other nonsense suppressor(see FIG. 1A). In addition, codons for natural amino acids could be used(e.g., valine). This resulted in a library with a primary sequencediversity with approximately 4×10⁶. The library was designed such thatmembers can contain 0 to 10 N methyl amino acids, with two being themost common. In addition to the N-methyl residues, this mutagenesisresulted in insertion of a number of natural sequences (FIG. 1A).Libraries were cyclized such that there was a mixture of linear andcyclic peptides, subjected to protease digestion, and selected forbinding to the target (FIG. 1B).

The selection procedure can be repeated until a sufficient degree ofbinding between the peptides and the target is shown. In one aspect,sufficient binding was shown after 5 rounds of selection and the poolwas subsequently sequenced. All of the 23 sequences found from thisselection contained at least 1 NMA. The average quantity of NMA perpeptide sequence increased in both the round 4 and round 5 pools withrespect to the round zero pool. One sequence, MFYAYEYAQWSK (SEQ ID NO:7) was found to dominate the pool. By pulldown, it was determined thatapproximately 35% of the sequences in the pool coded for this peptide.To the best of our knowledge, this is the first functional peptidederived by selection to incorporate unnatural amino acids for theenhancement of drug like characteristics. This peptide was attractivefor other reasons as well. It incorporated a core motif of YEY that issimilar to the core motif found in multiple other selections against Gαiproteins. The NMA residues are spaced apart, and predicted to provide asufficient window of protease resistance predicted by previouspredictive measures. There is also only a single lysine residue,indicating that there is only one possible cyclization. This moleculewas named SUPR for scanning unnatural protease resistant peptide.

There are significant changes from the parental molecule to the selectedmolecule. 8 of the 10 possible positions for amino acid changes showed achange in sequence. Some of these changes, such as PHE and THR incycGIBP to TYR and SER respectively, were conservative. However mayother, such as the VAL and GLY of cycGIBP to GLN and TRP respectively,show a significant change in side chain functionality that would nothave been predicted by analyzing sequences of Gαi1 binding peptides. Astructural comparison can be made by observing sequence changes asoutlined in FIG. 2.

The peptide was characterized by utilizing standard pull down assays.cycSUPR peptide was synthesized incorporating a biotin at theC-terminus. The peptide was immobilized on NeutrAvidin-agarose and usedto pull down radiolabeled protein. The efficiency of this pulldown wastested against multiple different Gα subunits. cycSUPR peptide retainedspecificity of its parent molecule as can be seen in FIG. 3. This wastrue both for specificity within the Gα family as well as statespecificity of Gαi1. In fact, there was a small enhancement ofspecificity for the target Gαi1 over a protein with nearly 85% sequencesimilarity, Gαi2. The ability to match target specificity is in directcontrast to results from chemical incorporation of N-methyls whichresulted in altered selectivity in 8 of 9 positions. The ability toretain or enhance selectivity for a desired target is of tremendousimportance for therapeutic and diagnostic development. This datasuggests that N-methyl incorporation into mRNA display will provide thatability.

Protease resistance enhancement was then tested. The simplest solutionfor enhancing protease resistance would be the removal of amino acidsrecognized by protease K and chymotrypsin. However, this peptide hasroughly as many theoretical digest sites as the parent molecule. Upondigestion with proteinase K and chymotrypsin, SUPR peptide has seven andfive theoretical digest sites, respectively. However, samples analyzedshowed only 1 digest site from proteinase K and two from chymotrypsin.Proteolytic digestion revealed that SUPR cyclic peptide wasapproximately 5200 fold more resistant to proteinase K and 200 fold moreresistant to chymotrypsin when compared to CycGIBP. Cyclization of SUPRenhanced protease resistance to both of these enzymes, suggesting acooperate effect between cyclization and N-methylation as may be notedin FIGS. 4A to 4D.

These enhancements translated to stability when subject to human serumas well. Human serum is composed of more than 1500 proteins and hundredsof proteases. This is a close in vitro mimic to the in vivo environmenta peptidic therapeutic would experience. Therefore for a peptide to beviable as a potential therapeutic, it must be stable to human serum.Current drugs, including peptidic drugs and monoclonal antibodies, showserum half-lives of days to weeks. LinGIBP and cycGIBP showed ahalf-life of approximately 0.14 hrs and 0.33 hrs respectively, far tooquick to be predicted to be a potential drug candidate. LinSUPR alsoexhibited poor stability, with a half-life of only 0.27 hrs. This islikely due to some sort of exopeptidase activity, since it is generallyknown in the field that N-terminal alkylation and C-terminal amidationwill help with stability. The incorporation of both cyclization andN-methylation found in cycSUPR peptide resulted in a very significantstability increase, generating a peptide with a 160 hr half-life inhuman serum shown in FIG. 4E. This data suggests that survivingchymotrypsin and proteinase K degradation is sufficient for conferringgeneral protease resistance because neither active chymotrypsin norproteinase K is found in human serum.

Another vital area for the in vivo processing of drugs is the liver. Allblood is eventually filtered through the liver, where various types ofenzymes functionalize drugs for the purpose of excretion. The largestand most active class of liver enzymes involved in this process are thecytochrome P450s. These enzymes are largely involved in the oxidation ofdrugs, and can be found on the extracellular surface of the liver. Humanliver microsomes are functional liver extract containing cytochromeP450s. Therefore half-life of modification of peptides by cytochromeP450s from exposure to human microsomes was examined. The results weresimilar to the human serum result in that both cyclization andN-methylation were necessary for significant enhancement of resistanceto modification. In fact, there was nearly an order of magnitudeincrease in stability from either cycGIBP or linSUPR (similarhalf-lives) to cycSUPR as shown in FIG. 4F. This is interesting becausethe trait of protease resistance, not resistance to cytochrome P450modification was a selection criteria. This suggests that theenhancements conferred by resistance to chymotrypsin and proteinase Kwill not only translate to general stability to proteases, but also toresistance to other classes of enzyme modification.

Crystal structures of serine proteases such as chymotrypsin andproteinase K show a hydrogen bonding network from the protease to thebackbone of their substrate at four amino acid positions. Applicantspostulated that N-methylation would result in a steric clash between thesubstrate and protease, thereby inhibiting the ability of the proteaseto bind to the peptide. In enzyme kinetic terms, this would result in anenhancement of Km. Additionally, several experiments have shown thatproteases such as chymotrypsin, will not cut all of their predicteddigest sites equally. It has long been observed that secondary structurewill have an effect on proteolysis of a protein. However, there are nowseveral independent examples of peptides with, with no predictedsecondary structure that have predicted protease digest sites that arenot proteolyzed. Applicants suspect that there may be some localsequence context that is inhibitor for proteolysis. This sequencecontext may either make it harder for the protease to bind its substrate(Km), or it may make it more difficult for the protease to performcatalysis on the substrate (Vmax). To investigate the origin of theseenhancements, Applicants conducted a basic kinetic study withchymotrypsin. Proteinase K digested GIBP too quickly for this type ofanalysis but could be utilized in other systems and with other peptides.What Applicants found was that SUPR peptide optimized both Km (bindingof enzyme to substrate), and Vmax (ability of the enzyme to process thesubstrate) were optimized for protease resistance relative to cycGIBP asmay be observed in FIG. 5.

Next the role of each of the N-methyl alanines was analyzedindependently. A traditional medicinal chemistry approach would suggestoptimizing each position in a stepwise manner. For example sequencemodifications, including N-methylation would be accomplished one aminoacid at a time, walking the peptide from low protease stability to highstability. Whether this is a viable approach for making a peptide likeSUPR was investigated. Towards that end, Applicants walked back fromSUPR peptide to something closer to cycGIBP, and to determine if therewas a gradual decrease in protease resistance. Towards that end, threepeptides were synthesized. Two of them contained only one NMA (alaninewas substituted for NMA), and the other had no NMA. It was hypothesizedthat if one could walk back to cycGIBP, then one would expect the lossof a single N-methyl to have some effect on reducing proteaseresistance, but to still be better than cycGIBP. One could also predictthat the double mutant, SUPR peptide sequence containing noN-methylations would exhibit similar properties to cycGIBP. The effectswere dramatic and surprising. Loss of a single NMA in both cyclic andlinear sequences resulted in protease resistance significantly worsethan that of the parent molecule GIBP. This was true for bothchymotrypsin digestion and proteinase K digestion as detailed inTable 1. In fact, the proteolysis was so efficient for chymotrypsin thatApplicants were not able to measure its half-life. This implies not onlythat there is a synergistic effect between the N-methylation but alsothat it would be extremely difficult to derive this peptide bytraditional stepwise optimization.

TABLE 1 Synergistic effect of N-methyl alanine. Loss of a singleN-methyl alanine results in peptide stability that is equal to, or lowerthan the stability of GIBP. Chymotrypsin Sequence Lin t_(1/2) Cyct_(1/2) MITWYEFVAGTK 0.33 min 2.1 min (SEQ ID NO: 19)

21 min 394 min

<0.2 min <0.2 min

<0.2 min <0.2 min MFYAYEYAQWSK <0.2 min <0.2 min (SEQ ID NO: 22)Proteinase K Sequence Lin t_(1/2) Cyc t_(1/2) MITWYEFVAGTK <0.2 min 1.0min (SEQ ID NO: 19)

2800 min 5300 min

<0.2 min 0.71 min

<0.2 min 0.77 min MFYAYEYAQWSK <0.2 min <0.2 min (SEQ ID NO: 22)

Experiment No. 2

This experiment shows application of the method of Experiment No. 1 tosynthesize peptides with pre-determined functionality. In addition,N-methyl norvaline was utilized and the starting peptide was selectedfrom an antibody loop region.

Every year 50,000 women are diagnosed with the highly lethal Her-2 (+)breast cancer. Herceptin (Trastuzumab) is the only FDA-approvedbiological therapy but costs $100,000 per year. This creates a financialbarrier to health care or a tremendous burden for those who choose topay for treatment. Furthermore, treatment is painful, inconvenient, andpresents an infection risk since it must be administered through largeIV injections. Herceptin sales generate ˜$5 billion annually for Roche,validating the size and importance of this market.

Her-2 (Human epithelial growth factor 2) is a receptor tyrosine kinasefound to be overexpressed in 20-30% of breast cancer patients. Lin, N.et al. (2007) Clinical Cancer Research, 13:1648. Her-2 is a uniquereceptor, having no known ligand, and functions by homo- andheterodimerization with other Her (1, 3 and 4) family members.Dimerization promotes the stimulation of cellular proliferation,invasion, and anti-apoptosis (Lin et al. (2007), supra.)

Clinically, the overexpression of Her-2 in breast cancer correlates to amore invasive disease with increased tumor growth, chemotherapyresistance, and significantly lower long term survival for patients.Sausville, E. A and Burger, A. M. (2006) Cancer Research, 66:3351. Everyyear, ˜50,000 women are diagnosed with this form of breast cancer.Monoclonal antibodies to the ectodomain of Her-2 have been shown to beclinically effective in limiting the growth of tumors in vivo. Herceptinwas one of the first examples of a FDA approved monoclonal antibody forthe treatment of cancer. However, as is typical with antibodytherapeutics, treatment is very expensive. Herceptin is typically dosedat 2 to 8 mg/kg costing patients up to $100,000 per year (Szabo, L.(2006) USA Today, available at the web address:www.usatoday.com/news/health/2006).

As with all antibody therapeutics, they are expensive to develop,control quality, produce, store, dose, not orally available, and theirsize limits their efficacy with solid tumors (Cho, M. J. and Juliano, R.(1996) Trends in Biotechnology, 14:153). Stabilized peptide therapeuticscould greatly benefit patients with this disease by increasingaccessibility to treatment by dramatically reducing cost and providing apotentially oral route to administration.

THG73 tRNA Preparation.

The method described in Example 1 was followed.

N-methyl Norvaline-tRNA.

Norvaline is an amino acid with a straight 3 carbon side chain, andreasonably isosteric for methionine. N-methyl norvaline translates moreefficiently than N-methyl alanine. When translating the sequence MFXFF(SEQ ID NO: 23), where X is the n-methyl amino acid, N-methyl norvalineshows twice the suppression efficiency (the ratio of translationefficiency with tRNA-N-methyl amino acid to translation withouttRNA-N-methyl amino acid), as can be seen in FIG. 10. The synthesis ofN-methyl, N-nitroveratrylcarbonyl norvaline cyanomethyl ester wascarried out according to methods known in the art and publishedprotocol. The final product was purified by silica gel chromatography in3:1 EtOAc/hexanes. Yield 187.5 mg (24%). The synthesis of N-methyl,N-nitroveratrylcarbonyl alanine-dCA was carried out according toprevious protocol. Yield % 0.5 mg (2.5%). Following ligation to THG-73tRNA, deprotection of the nitroveratryloxycarbonyl group was effected byphotolysis with a xenon lamp equipped with a 315-nm cutoff filter, andthe N-methyl norvaline tRNA was immediately added to the translationreaction.

Synthesis of N-Methyl Scanning Library.

Two single-stranded DNA templates were ordered from Integrated DNATechnologies. The sequences were 5′-GGG ACA ATT ACT ATT TAC AAT TAC AATGNN SKR SKA KKR KTW STA KKM SNN SAA AAG TAG TGG TAG CAG CGA TTA CA-3′(SEQ ID NO: 24) and 5′-GGG ACA AAT ACT ATT TAC AAT TAC AAT GNN SYM KYAKKM KYA STW KNN SAA AAG TAG TGG TAG CAG CGA TTA CA-3′(SEQ ID NO: 25)using machine mixing for variable positions (W=A or T, K=G or T, M=A orC, R=G or A, S=G or C, Y=T or C). The double stranded library wasproduced and amplified by performing 6 cycles of PCR on 1 pmole of PAGEgel purified single-stranded library. PCR was performed under standardconditions utilizing the primers Gen-FP (5′-TAA TAC GAC TCA CTA TAG GGACAA TTA CTA TTT ACA ATT ACA-3′ (SEQ ID NO: 11)) and the reverse primer(5′-TGT AAT CGC TGC TAC CAC TAC TTT T-3′ (SEQ ID NO: 26))

The Round 0 mRNA pool was generated by T7 runoff transcription andpurified by urea-PAGE. The purified mRNA was ligated to F30P(5=−dA21[C9]3dAdCdC-P (SEQ ID NO: 74); C9% tri-(ethylene glycol)phosphate (Glen Research), P % puromycin (Glen Research)), via anoligonucleotide splint (5′-TTT TTT TTT TTT TTG TAA TCG CTG C-3′ (SEQ IDNO: 27)). Ligation was urea-PAGE purified and quantified via absorbanceat 260.

Translation and Cyclization.

The method described in Example 1 was followed.

Selection.

Obtaining target protein for this selection step was a technicalchallenge. Her-2 is a multidomain protein, and the peptides had totarget the ectodomain and not the intracellular kinase domain.Additionally, Her-2 is heavily post-translationally modified. Expressingand purifying functional Her-2 with the correct post-translationalmodifications would be difficult. However, several immortalized celllines have been derived from breast cancer patients with Her-2 heavilyoverexpressed and since they are human cell lines, should have all thepost-translational modifications present in a Her-2 positive cancerpatient. Following ethanol precipitation of the round 0 DSG treatedfusions, the pellet was dissolved in 100 μL of dH₂O and reversetranscribed with Superscript II under standard conditions. Followingreverse transcription, the library was diluted 2 fold into 50 mMpotassium phosphate buffer (pH=8.0). Proteolysis became more stringentas the selection continued. All proteolysis used immobilized proteasespurchased from Sigma. Round 0 fusions were subject to 0.1 mgs ofproteinase K for 30 sec. Round 1 was proteolyzed with 1 mg of proteinaseK for 30 sec. Rounds 2 and 3 fusions were proteolyzed with 1 mg ofproteinase K for 5 min. Finally, round 4 fusions were proteolyzed with 1mg of proteinase K, 1 mg of chymotrypsin, and 1 mg of trypsin for 5 min.All proteolysis was performed at room temperature. Proteases wereremoved by spin filtration.

Two days prior to selection 150,000 SKBR-3 cells per well were seededinto 2 wells of a 96 well plate and grown under standard cell cultureconditions (D-10 media, 5% CO₂, 37° C.). SKBR-3 cells were incubatedwith proteolyzed fusions that had been diluted to 400 μL in D-10 media(200 μL used per well). After 1 hr at room temp fusions were removed andcells were washed 4 times with D-10 media. After washings, cells wereproteolyzed for 20 min at room temp. using proteinase K (0.5 mgs/well).Fusions were removed from cells and protease by spin-x filtration. TheHer-2 libraries were regenerated by a 400 μL PCR using the “PhusionBlood Direct PCR Kit” (New England Biolabs), following manufacturesinstructions. PCR amplification of the eluted library members wascarried until band was observed on a 4% agarose gel (15-20 cycles).

The round 5 pool was amplified by PCR using taq polymerase and subclonedinto the TOPO-TA vector (Invitrogen) followed by transformation intoTOP10 competent cells (Invitrogen). Individual clones were sequenced(Laragen).

Cell Culture.

SKBR-3 cells were purchased from the ATCC. DMEM and FBS were purchasedfrom GIBCO. Cells were cultured under standard conditions using D-10media.

Cell Proliferation.

Proliferation was measured by standard BrdU cell proliferation assay(Calbiochem). Cell lines were plated in 96-well plates (20,000cells/well) in D-10 media with indicated amount of peptide with 2% DMSO,and incubated overnight. The BrdU compound was given to the cells for 2hrs. Following the manufacturer's protocol, samples were analyzed by UVabsorbance measurements at 450 nm. Peptide signal was normalized tocells incubated with 2% DMSO but no peptide. IC50 data was generated byfitting the data to a drug response equation (Log(drug) vs. Response,GraphPad Prism 5.0).

In Vivo Studies.

NCr homozygous athymic (nude) mice (six to eight weeks-old) werepurchased from the National Cancer Institute. An aliquot of 2×10⁶ SKBR-3cells were suspended in 200 ml of PBS and injected subdermally in theright thigh of each animal. Treatment began 7 days after inoculation.Peptide 1 and Peptide D were coadministered at a total peptideconcentration of 7 mg/kg three times a week by IV injection. Tumorgrowth was monitored weekly for four weeks. Tumor volume measurementswere my following standard protocols using an electronic caliper.

Peptide Synthesis.

All solvents were purchased from Sigma. All other peptides weresynthesized using fmoc-Gly-Wang resin (250 mgs, 0.15 mmoles) unlessotherwise specified. Standard couplings were carried out with 5 eq. ofmonomer on a PS-3 automated peptide synthesizer (Protein Technologies).Fmoc deprotection was carried out with 20% methyl piperidine at roomtemperature for 10 min. After the addition of the N-terminal amino acid,peptides were capped with glutaric anhydride. Following N-terminalcapping, lysine(mmt) was selectively deprotected with 3% DCM, 1.5% TIS,and 1.5% EDT in DCM for 1 hr at RT. After washing resin with NMP,cyclization on resin was accomplished by the addition of HATU (5 eq) andDIEA (10 eq) and rotating for 1 hr at RT. Following cyclization,deprotection, cleavage with 95% TFA, filtration and ether extraction,the crude product was purified on a Vydac C-18 reverse phase columnusing gradient elution (0% B for 5 min, 10-50% B in 40 min. Solvent A:H₂O with 0.1% TFA, Solvent B: CH₃CN with 0.035% TFA. Lyophilized solidwas reconstituted in DMSO and quantitated by absorbance at 280 nm(ε280=9970 L mol-1 cm-1). Yield=10-25%.

Human Serum Digests.

Delipidated/lyophilized human serum was purchased from Thermo Scientificand reconstituted as per manufacturer's instructions. 250 nmoles ofpeptide in 50 μL of sodium phosphate buffer (pH=8.0) with 10% DMSO wasadded to 1 mL of reconstituted serum and incubated at 37° C. 100 μLaliquots were taken at various time points and quenched in 300 μL ofacetonitrile. Samples were spun down and decanted to remove precipitatefollowed by dilution in water to 1.5 mLs. Samples were injected onto aC-18 reverse phase column and separated by gradient elution (15-90% B in25 min. Solvent A: 0.1% TFA in water. Solvent B: CH₃CN (0.05% TFA). Thearea under the starting material peak was quantitated using the 32 KaratGold Software package (Beckman). The plotted values represent the meanof two experimental values, and the error bars represent the standarderror of the mean. The graph was generated by fitting the data to a onephase exponential decay equation (GraphPad Prism 5.0).

Discussion

Two peptides were derived from examining the antibody loops involved intarget binding. ANHP is a peptide derived from the Herceptin loop,(Park, B.-W. et al. (2000) Nat Biotech, 18:194) and HRAP was derivedfrom Omnitarg (Nakajima, H. et al. (2008) Breast Cancer 15:65). Bothexhibit in vitro function at high micromolar concentrations. From thesepeptides, two separate cyclic peptide libraries were developed. Aflanking randomized residue was placed on either side of the leadpeptide sequence which is illustrated in FIG. 8.

The two resulting peptide libraries were of different lengths (MX8K forthe Herceptin based library, and MX₇K for the Omnitarg based library asdepicted in FIG. 9) which allowed tracking to determine which peptidesare derived from which original family of ligands to choose forcoadministration. The two libraries were mixed after round 0 PCR inequal proportion and carried through the selection together, giving atotal diversity of 21 million unique DNA sequences.

Applicants used fluorescein labeled HRAP peptide to quantitate Her-2expression. Peptide was incubated with cells, and after washing thecells were trypsinized and analyzed on a fluorometer. Human cells highlyexpressing Her-2 will have 2 million or more copies on their surface.Applicants found that there were approximately 4.5 million copies ofHer-2 expressed per cell. 200,000 cells would contain approximately 1.5pmole of target, which is in the range necessary for targeting by mRNAdisplay.

Selection was conducted under similar conditions as described inExample 1. After 4 rounds of selection, pool 5 was sequenced andresulting sequences were separated into their original families. 5members from the Omnitarg (named 1 through 5, 1-MAVYVHYHK (SEQ ID NO:28), 2-MSYHYVVPK (SEQ ID NO: 29), 3-MLSYSHVQK (SEQ ID NO: 30),4-MEYVSYVAK (SEQ ID NO: 31), 5-MRHQEVLLK (SEQ ID NO: 32) where V isN-methyl norvaline), family and 5 members from the Herceptin family(named A through E, A-MQYDEYVDSK (SEQ ID NO: 33), B-MLWDEYVACK (SEQ IDNO: 34), C-MMWVEFYSLK (SEQ ID NO: 35), D-MVCVVLYDDK (SEQ ID NO: 36),E-MVCEYYVYSK (SEQ ID NO: 37) where V is N-methyl norvaline) were chosenfor initial screening. Cyclized fusions were tested for binding beforeand after proteolysis. The top binding peptide in each family was alsothe most protease resistant. These peptides, peptides 1 and D, werechosen for further characterization. Comparing the N-methylatedmolecules with the parental molecules showed significant changes. Inpeptide D, 5 of the 6 original residues had changed. One of themutations was conservative, however the others would have been difficultto predict by rational design. In peptide 1 of the 5 positions changed,however the changes showed some conservation of the originalfunctionality. For example, PHE changed to TYR and PRO changed toN-methyl norvaline. In examining the structures it is evident that thereare significant chemical similarities between these peptides. However,both in the case of peptide 1 and D, the overall peptide structure issignificantly different from the parental sequence. This data, suggeststhat their development by traditional medicinal chemistry would beextremely difficult. A schematic showing the structural changes can beseen in FIG. 12.

The peptides were then screened for function using a standard in vitroassays used to test efficacy of Her-2 targeting molecules. The assayinvolved analyzing a molecule's ability to inhibit proliferation in cellculture. A widely used kit is the BrdU cell proliferation assay. In thisassay, cells are incubated with the molecule of interest before havingtheir media supplemented with a bromodeoxyuridine. Bromodeoxyuridine isincorporated into the DNA of proliferating cells. DNA with this modifiedbase is recognized with an antibody, and quantitated by ELISA.Applicants tested the peptides and compared them with their parentalmolecules. There were dramatic improvements in efficacy for bothpeptides. HRAP had an EC₅₀ of 230 μM in comparison to peptide 1's EC₅₀of 520 nM, a nearly 450 fold improvement. ANHP had an EC₅₀ of 67 μM⁷ incomparison to peptide D's value of 640 nM, nearly a 110 fold enhancement(see Table 2).

Applicants then added the peptides in equal concentration to cellculture and performed the BrdU cell proliferation assay. Applicantsfound an enhancement resulting in an EC₅₀ corresponding to a totalpeptide concentration of 15 nM. This corresponds to a 40 foldenhancement over peptide 1 and a 35 fold enhancement over peptide D whenadministered individually (Table 1). This suggests that peptide 1 andpeptide D are functioning cooperatively.

TABLE 2 Peptide IC50 HRAP 230000 nM   Peptide 1 520 nM AHNP 67000 nM Peptide D 640 nM Peptide D + 1  15 nM Table 2: Dramatic improvement inthe inhibition of proliferation of Her-2 positive cells based on BrdUincorporation. Peptide 1 and Peptide D show approximately a 440 and 100fold improvement with respect to their parental molecules.Coadministration of Peptide 1 and Peptide D shows approximately a 35 and43 fold improvement over individual administration respectively.

Another measure of the effectiveness of these peptides is their maximalinhibition on cellular proliferation. That is, at concentrationssignificantly above the EC₅₀, how much slower is the proliferation ofSKBR-3 cells. SKBR-3 cells treated with Herceptin proliferate at 75% therate of untreated cells (25% inhibition). Peptide 1 and peptide D showinhibitions of 75% and 56% proliferation respectively (25% and 44%inhibition respectively) (see FIG. 13). HeLa cells were used as acontrol because they do not overexpress Her-2 and are not responsive toHer-2 targeting treatments. As expected, HeLa cells showed no inhibitionin their ability to proliferate. If peptide 1 and peptide D function ina cooperative manner producing a multiplicative effect on inhibitionApplicants would expect coadministration to exhibit an inhibition of 42%proliferation (58% inhibition). In fact the value was 39% proliferation(61% inhibition), within error of this prediction (see FIG. 13).

Finally Applicants wanted to determine if selected peptides couldillustrate in vivo function. Literature shows that xenografts treatedwith Herceptin were characterized by a slower tumor growth in comparisonto mice without treatment. Nude mice were inoculated with SKBR-3 cells.Mice were treated 3 times weekly for 4 weeks with intravenous injectionsof peptide 1 and peptide D with a total peptide concentration of 7mg/kg. Mice treated with peptide showed either a halt in the progressionof tumor growth, or a decrease in tumor volume. This result exceedspublished results for Herceptin in this model system. See FIG. 14.

Even with advances in biotechnology, many proteins are difficult topurify in their functional state. By successfully targeting proteinexpressed on a human cancer cell, Applicants have derived a new path toobtaining targets for mRNA display. This also includes the creation ofligands to many proteins that were too technically difficult to targetin the past. There are also implications to personalized medicine. Thetargeted cells in this selection, SKBR-3 cells, came from a breastcancer patient. An obvious extension of this technology would be todevelop it to the point where a biopsy may be taken from a cancerpatient, and those cells used as a target in this type of mRNA displayto derive a truly personalized treatment.

With the targeting of Her-2, Applicants have shown that through thecombination of cyclization and N-methyl amino acid incorporation intomRNA display Applicants are able to derive bioavailable peptide ligands.These ligands show in vivo stability sufficient for drug like function.These enhancements translated to peptides that could show high levels ofin vivo efficacy. Additionally, the process of lead development wasfast. Target selection to lead characterization was 3 months. In vivoefficacy was shown in a total of 5 months. This data validates this mRNAdisplay method as a rapid platform technology able to efficientlydevelop highly stable, efficacious peptidic ligands for the purpose ofpeptidomimetic development in disease treatment. These Her-2 inhibitingpeptides are merely the first of many in a new class of peptidomimeticsable to be developed as highly effective treatments for disease.

Experiment No. 3

This example illustrates modification of the peptides for oralbioavailability.

Cellular Uptake Assay.

Fluorescein labeled peptide added to approximately 1.5×10⁵ HeLa cells in200 μLs of DMEM growth media with 5% FBS such that the final peptideconcentration is 10 μM. Cells were incubated overnight at 37° C. with 5%CO₂. Cells were washed 4 times with PBS, trypsonized, and bufferexchanged to PBS. The cells were analyzed by using fluorescent flowcytometry and dead cells were excluded from the analysis. The datapresented are the mean fluorescent signal for the 5,000 cells collected.

Confocal Microscopy.

Approximately 1.5×10⁵ HeLa cells were plated on a 96 well plate. Cellswere incubated with 10 μM fluorescein labeled peptide in DMEM media with5% FBS and 2% DMSO overnight. The cells were washed with PBS andobserved with an inverted microscope using a 60× objective. 490 nm lightwas used to excite fluorescein and a 520 emission filter was used forobservation of the green emission. When comparing the uptake or activityof the peptides the imaging conditions (such as photomultipliergain/offset, laser intensities and confocal aperture size) were keptconstant for the observation of the different conjugates, so that theintensities represent the true differences in uptake/activity.

Oral Bioavailability.

The following peptides were synthesized for oral administration:MFYVYEYVQWSKK(FAM) (SEQ ID NO: 14), MITWYEFVAGTKK(FAM) (SEQ ID NO: 16),MFYVYEYVQWSKK(FAM)DK(biotin) (SEQ ID NO: 38), andMFYVYEYVQWSKK(FAM)DK(Palmitoleic acid) (SEQ ID NO: 15). All peptideswere cyclized from the side chain to lysine to the N-terminus aspreviously described in Millward, S. W. et al. (2007) ACS Chem. Biol.2:625. Anesthetized C57BL6 mice had peptide administered at a dose of 10mgs/Kg by oral gavage. After administration, mice were removed fromisoflurane. At various time points, blood samples were taken by orbitalbleeding as previously described. Peaks were normalized to a 20 minutetime point from mice that had peptide administered by IV injection. Areaunder the curve was analyzed by Graphpad 5.0.

Peptide Synthesis.

All solvents were purchased from Sigma. Peptides were synthesized bymanual solid-phase peptide synthesis using rink amide am resin,following standard protocols. Standard couplings were carried out withmonomer (5 equiv; Novabiochem) in HATU (2 mL, 0.6 mmol; Novabiochem),HOAt (1.2 mmol; Genescript) in DMF with DIEA (1.8 mmol) at roomtemperature for 20 min. Coupling to an N-methyl amino acid followed thesame procedure with a 30 min coupling time. Fmoc deprotection wascarried out with 20% piperidine (Anaspec) at room temperature for 20min. Following, deprotection, cleavage with 95% TFA, filtration andether extraction, the crude product was purified on a Vydac C-18reversed-phase column using gradient elution (0% B for 5 min, 10-50% Bin 40 min. Solvent A: H2O with 0.1% TFA, Solvent B: CH3CN with 0.035%TFA). Lyophilized solid was reconstituted in DMSO and quantitated byabsorbance at 280 nm. Yield=10-25%.

N terminal specific biotinyation with reducible biotin was accomplishedby reacting peptides with NHS-biotin (Pierce) following manufacturer'sprotocols. Cyclosporine was purchased from Sigma Aldrich. DNA waspurchased from IDT DNA.

PAMPA.

PAMPA assays were set up following manufacturer's instructions. Briefly,1% phosphatidyl choline (Sigma) was dissolved in dodecane (Sigma) at 65°C. for 3 min. The membrane was applied to each well as permanufacturer's instructions. Peptides 1-10 and 1-10B (see Table 3, 15 μLof 4 mM peptide in DMSO) were diluted to 300 μL in PBS pH 7.4. 100 μL ofsample was put into each donor well. The acceptor wells contained 300 μLof a 5% DMSO in PBS solution. Passive diffusion was allowed to occur atroom temperature for 24 hrs. At this time, samples were run on a HPLCVydac C-18 reversed-phase column using gradient elution (0% B for 5 min,10-50% B in 40 min. Solvent A: H₂O with 0.1% TFA, Solvent B: CH₃CN with0.035% TFA). Absorbance at 215 nm was monitored. The area under thepeptide peak was integrated. Due to cyclosporine A's low absorbancereading, multiple wells acceptor solutions had to be combined.

Peptides 1RB, 8RB, and 10RB (sequences are provided in Table 3) followedthe changes. The acceptor well contained 10 mM DTT as a reducing agent.Data was collected after 6, 12 and 24 hrs. Absorbance at 280 nm wasanalyzed. The following formula was used to calculate Log Pe:

${\log \; P_{E}} = {\log \left( {C - {\ln \left( {1 - \frac{\lbrack{drug}\rbrack {acceptor}}{\lbrack{drug}\rbrack {equilibrum}}} \right)}} \right)}$$C = \left( \frac{{Vd} \times {Va}}{\left( {{Vd} + {Va}} \right){Area} \times {time}} \right)$

Membrane Localization.

Membrane was set up as described above. Donor and acceptor wells hadpeptides at 0.2 mM. Peptide concentration in the donor well wasquantitated by absorbance at 280 nM at times 0 and 15 min. Care wastaken not to disrupt the membrane when samples were removed.

Insulin Biotinylation and Characterization.

0.25 mmoles of human insulin was reconstituted in 1 mL 50 mM sodiumphosphate buffer (pH=8.0). To this was added 0.5 mmoles of sulfo-NHSbiotin (pierce). The reaction was allowed to proceed for 1 hr at roomtemp before HPLC purification on a on a Vydac C-18 reverse phase columnusing gradient elution (0% B for 5 min, 10-50% B in 40 min. Solvent A:H₂O with 0.1% TFA, Solvent B: CH₃CN with 0.035% TFA. Peaks were analyzedby MALDI-TOF. Two peaks corresponded with single biotinylations and weretested for efficacy in vivo. The second peak was determined to be moreefficacious and used for further characterization. To determine the siteof biotinylation, purified singly biotinylated insulin was digested with0.1 mg of immobilized chymotrypsin agarose (sigma) for 1 min at roomtemp. Reaction was stopped by filtration. MALDI-TOF analysis was used todetermine that biotinylation occurred on the N-terminus of the B-chain.

In Vivo Effect of Peptides In Vivo.

Experimental procedure followed previously published protocols withslight modification, see e.g., (McKern, N. M., et al. (2006) Nature443:218; Schïffer, L., et al (2003) PNAS 100:4435). Briefly, C57BL6 mice(4 in each group) fasted overnight and were anesthetized usingisoflurane prior to taking blood samples by orbital bleeding. Mice weredosed with insulin or biotinylated insulin at a concentration of 3.5nmol/kg by IV injection into the tail vein. For oral bioavailability,insulin was dosed at 1.75 and 7 mol/kg and administered by oral gavage.In all cases, insulin was in 150 μL of 50 mM sodium phosphate buffer(pH=8.0). Blood was analyzed on a standard Clarity Plus blood glucosemeter (VWR). Readings were normalized to a T=0 blood sugar measurement,and analyzed on Graphpad 5.0.

A number of limited predictive models also exist that are more closelyapplicable to the development of peptide ligands. First analysis byVeber et al., (Veber, D. F. et al. (2002) Journal of Medicinal Chemistry45:2615) indicates that oral availability correlates with having 10 orfewer rotatable bonds and argues that large cyclic molecules may bequite cell-permeable. Second, experimental work by Lokey and coworkersindicates that cyclizing peptides can dramatically improve their passivemembrane permeability by 10- to 100-fold, generating molecules withsimilar membrane crossing ability as cyclosporine. Rezai, T. et al.(2006) Am. Chem. Soc. 128:2510. However the generality of these resultshas yet to be determined, and there are a number of examples wherecyclization does not seem to help permeability. Kwon, Y.-U. and Kodadek,T. (2007) Chemistry & Biology 14:671.

Work from both the Kuliopulos and Smrcka labs indicates that attaching along chain fatty acid (palmitic C16, or myristic C14) enablespeptide-fatty acid conjugates added to cells to gain access to thecytosol. (Veber, D. F. et al. (2002) Journal of Medicinal Chemistry45:2615; Kwon, Y.-U.; Kodadek, T. (2007) Chemistry & Biology 14:671).The mechanism of this has not been studied in detail, but it appears thefatty acids incorporate into the outer leaflet, followed by transfer tothe inner leaflet. Further studies showed that these compounds cross theblood brain barrier. (Zhuang, Z. P. et al. (2001) Journal of MedicinalChemistry 44:1905). Finally, Meade and coworkers have shown that astilbene derivative, (ADMS), could ferry a gadolinium chelate acrosscellular membranes for the purpose of serving as an intracellularcontrast agent. Baumann, K. et al. (1994) Protein Sci 3:750). In allcases, the transfer mechanism seems to be passive and implies thatattaching hydrophobic or amphiphilic molecules to peptides provide afacile route for them to cross cellular membranes. It also seems likelythat the more hydrophobic moieties (palmitic and myristic acid) keeptheir attached peptides associated with the membrane, similar toproteins modified in this fashion (Wadia, J. S. et al. (2004) Nat Med.10:310) resulting in lower efficiencies of passive transfer acrossmembranes.

There are only a few other oral insulin formulation in clinical trials.Most of these formulations focus on encapsulation of insulin to allowits transport through the stomach and release in the intestines. Thesetechnologies are difficult to perfect, require higher dosing of insulin,and require higher dosing in preclinical studies with suggests that theywill be significantly more costly than Applicants product. Furtado, S.,et al. (2008) International Journal of Pharmaceutics 347:149; Sonaje,K., et al. (2009) Biomaterials 30:2329; Yin, L., et al. (2009)Biomaterials 30:5691. Additionally, since none are yet FDA approved,Applicants have yet to see if this new technology will be free oftoxicity issues.

A GLP-1 analog appended to a fatty acid is also being developed by NovoNordisk for oral delivery. GLP-1 simulates the release of insulin in thepancreas of those with elevated blood sugar. Therefore this treatmentwill only be ineffective for type I diabetics who can still secretinsulin or who are not extremely sensitive to insulin (advanced type IIpatients). Additionally, the use of fatty acids for oral uptake has beenassociated with irritation of the intestines. Iyer, H., et al. (2010)Obesity and Metabolism 12:179 and Runge, S. (2008) 283:11340.

The biotinylated insulin of this disclosure has significant advantagesover all existing technologies. It is useful for both type I and type IIdiabetics, simple to synthesize, and cheap to manufacture. Since biotinis a vitamin (vitamin B7), and has undetectable toxicity, Applicantscompounds should be free of toxicity issues. Additionally, Applicantsdata shows that biotin is more efficient at delivering cargo across alipid bilayer as is necessary to target intracellular drug targets.

Applicants employed a parallel artificial membrane permeability assay(PAMPA) that will allow for the rapid screening of peptide passivediffusion across a phospholipid bilayer. (Schmidt, et al. (2003) J.Millipore Corporation Application Note AN1729EN00.). In this assay, twochambers are separated by a phospholipid bilayer supported on an inertmembrane. Applicants chose to initially focus Applicants examination onpeptide ligands developed to bind with high affinity to theheterotrimeric G-proteins Gαi1*GDP and Gα12*GDP. Ja, W. W., et al.(2004) Biochemistry 43:9265; Millward, S. W., et al. (2007) ACS Chem.Biol. 2:625; Fiacco, S. et al. (2008) ChemBioChem 9:2200. These ligandsincluded variations in sequence, size, overall charge, and polarity aswell as comparing linear and cyclic peptides, end and biotinylation.Many of the unmodified peptides were poor at crossing the phospholipidbilayer (Table 2). Two notable exceptions were peptide 1 and 3, whichexhibited permeability similar to that of cyclosporine. According toclassical methods of increasing permeability. Pinski, C. A., et al.(2001) Advanced Drug Delivery Reviews 46:3. Applicants postulated thatthe removal of hydrogen bond donors by N-methylation should furtherincrease the permeability of this peptide. Peptides 2 and 9 experienceda significant increase in permeability. This may be due to the removalof a positive charge at their terminus. This data suggests thatmodification at the termini of a peptide may be generally beneficial.

Recently there has been some controversy as to the effect of cyclizationon membrane permeability. Cyclic peptides are thought to benefit by anenhanced ability to shield their amide protons from the hydrophobicregion of a bilayer by more easily forming intramolecular hydrogenbonds. Rezai, T., et al. (2006) Am. Chem. Soc. 128:2510. However peptidecyclization has not always led to enhanced membrane permeability. Kwon,Y.-U. and Kodadek, T. (2007) Chemistry & Biology 14:671. In theexperiment, compound 7 was cyclized from the N-terminus to the sidechain of Lys. This cyclization route also removes two positive charges,which would be predicted to further benefit permeability. In thesefindings, cyclization did not enhance passive diffusion, and was in factslightly detrimental to the process.

Previous work has demonstrated that tethering peptide to fatty acids, orstilbene derivatives will allow peptides to access the cystolic side ofa cellular membrane. Covic, L., et al. (2002) Nat Med. 8:1161; Endres,P. J., et al. (2006) Molecular Imaging 4:485; and Goubaeva, F., et al.J. Biol. Chem. 278:19634. The mechanism has not been studied in detail,but the modification is thought to function by localizing the peptide tothe outer portion of the bilayer, followed by a transfer to the innersurface of the membrane. Applicants' aim was to find a nontoxic moleculethat could be conjugated to a peptide that would be hydrophobic enoughto enhance the localization of Applicants peptide to the phospholipidbilayer, without being so hydrophobic that it would not dissociate fromthe membrane. To that end, Applicants conjugated peptides to biotin, andexamined their permeability. A dramatic increase of up to 2 orders ofmagnitude in the Log Pe of peptides N- or C-terminally labeled withbiotin was observed. This provided five peptides with Log Pe valuessimilar to cyclosporine. It may also be noted, that this conjugationseemed to act as a general enhancer for peptide delivery. Enhancementsoccurred in spite of peptide sequence variation, overall charge,backbone modification, or length. Biotinylation of the side chain of Lysof peptide 9 did not enhance membrane permeability, suggesting that theenhanced permeability was not simply a result of increasedhydrophobicity or removal of a charge from the peptide. Taken incombination with the N-methyl data, this suggests that N- or C-terminalpositions may be optimal for modification.

TABLE 3 Log Pe of Log Pe of peptide Peptide Sequence peptide(biotinylated)  1 DKLYWWEFL* −7.1 ± −0.2 −6.4 ± −0.2 (SEQ ID NO: 39)  2Ac(N-MeD)KLYWWEFL* −6.1 ± −0.1 ND (SEQ ID NO: 40)  3 NNNNNDKLYWWEFL*−6.6 ± −0.2 ND (SEQ ID NO: 41)  4 NNNNNDK(N-MeL)YWWEFL* >−9.0 ND(SEQ ID NO: 42)  5 NNNNNDKL(N-MeY)WWEFL* >−9.0 −7.4 ± −0.1(SEQ ID NO: 43)  6 NNNNNDKLY(N-MeW)WEFL* >−9.0 −6.6 ± −0.1(SEQ ID NO: 44)  7 MITWYEFVAGTK^(†) −7.8 ± −0.1 −6.6 ± −0.1(SEQ ID NO: 45)  8 Cyclo-MITWYEFVAGTK^(†) −8.2 ± −0.2 −6.5 ± −0.0(SEQ ID NO: 46)  9 (N-MeNva)ITWYEFVAGTK^(‡) −7.0 ± −0.1 −7.4 ± −0.1(SEQ ID NO: 47) 10 MSQTKRLDDQLYWWEYL* −8.4 ± −0.1 −6.2 ± −0.0(SEQ ID NO: 48) 11 MRLVWIVRSRHFGPRLRMAK^(‡) ND −6.0 ± −0.1(SEQ ID NO: 49) Cyclosporin −6.2 ± −0.2 NA DNA >−9.0 >−9.0 Table 3:Membrane permeability is enhanced by biotin conjugation. Valuescalculated using PAMPA as outlined in Morris, M. C. et al. (2001) Nat.Biotech, 19: 1173. Biotin-labeled peptides are designated by a Bfollowing their number. Peptides 7, 8, 9, and 11 are amidated.*N-terminally biotin-labeled. ^(†)C-terminally biotin-labeled.^(‡)biotin-labeled on the side chain of Lys.

In order to study the mechanism of this enhanced permeation in moredetail, Applicants quantitated the biotin-peptide conjugate's membranelocalization. Peptides 1B, 6B, 7B, 8B and 10B exhibited membranelocalization of 8.4 to 14% respectively. Their nonbiotinylatedcounterparts, peptides 1, 6, 7, 8, and 10, localized 0 to 4.2%respectively. This suggests that the enhancement of membranelocalization facilitated peptide delivery.

There seemed to be a maximum permeability coefficient of about −6.0,irrespective of the peptides initial permeability. Applicants'hypothesis was that a slow step in this process was now the dislocationfrom the inner membrane to the acceptor well. Additionally, Applicants'permeation process was reversible. A biotin-labeled peptide couldlocalize to the membrane from the acceptor well, flip, and release intothe donor well. Applicants addressed this by creating a biotin conjugatewith a disulfide that would be labile intracellularly, but notextracellularly. This would expedite dislocation from the innermembrane, and create an irreversible delivery mechanism, trapping thepeptide inside the acceptor well.

In pursuit of this aim, Applicants conjugated peptides 1, 8 and 10 tobiotin containing a reducible disulfide. The PAMPA assay was set up sothat conditions in the donor well were oxidizing, and the acceptor werereducing. Log Pe significantly increased in all three cases. Incomparison to their nonlabile reducible counterparts peptide 1Bexhibited an increase in Log Pe from −6.4 to −4.9, peptide 8 increasedfrom −6.5 to −5.9, and peptide 10 increased from −6.2 to −4.8. This is a16-fold increase in peptide delivery for peptide 10. Applicants alsosought to determine if replacing biotin with a more hydrophobic moleculewould further enhance delivery. Initial attempts employed cholesterol;however peptide-cholesterol conjugates would aggregate under conditionsrequired for PAMPA. Lauric acid, a 12 carbon saturated fatty acid, wasalso employed and exhibited enhanced membrane association as compared tobiotin (27% vs. 14%). However these conjugates were not able to deliverpeptide cargo as efficiently, resulting in a Log Pe of −6.8 incomparison to biotin's log Pe of −4.8.

Next Applicants sought out to determine whether this was a viable methodof delivering peptide cargo in cell culture. If this were possible, itwould allow us to develop ligands to target intracellular proteins.Peptide 1 was labeled with a fluorescein at the N-terminus, and areducible biotin at the C-terminus as depicted in FIG. 17. Uptakestudies were performed by incubating peptide overnight in human cervicalcancer cells (HeLa), and mouse fibroblasts (3T3). Confocal analysisshows efficient delivery of peptide into both cell lines for peptidecontaining the biotin modification as can be noted in FIG. 17. However,cells incubated with protein without biotinylation did not showfluorescence. It also may be noted that the peptide has gained access tothe nucleus, suggesting that it may be cytoplasmic.

Applicants wanted to determine whether active transport played a role inbiotin mediated peptide uptake. 3T3 cells were incubated with adetran-rhotamine compound to label endocytotic vesicles. Significantcolocalization was noticed between Applicants' peptide and the dextranrhotamine as can be noted in FIG. 18. The experiment was repeated in thepresence of the dynamin dependent endocytotic inhibitor, dynasore.Uptake of Applicants' peptide still occurred, even in the absence ofinternalized dextran-rhotamine. Although active transport may bebeneficial for internalization of biotin shuttling, it seems not to benecessary.

Flow cytometry experiments were subsequently employed to compare biotinmediated delivery to those of cationic peptides. Truncations of HIV TATprotein has been shown to be capable of delivering cargo to cells.Wadia, J. S., (2004) Nat. Med. 10:310. Additionally polyargininesequences are thought to delivery cargo via a similar mechanism. Tocompare their efficiencies to biotin shuttling, two additional variantsof peptide 1 were synthesized. One contained an N-terminal TAT₍₄₇₋₅₇₎sequence, and the other had 9 N-terminal Arg residues. All peptides hada fluorescein at the N-terminus. Flow cytometry was employed toquantitate uptake efficiency. FIG. 19 show that biotin mediated deliverand disulfide release was 1.6 fold as efficient as TAT under standardconditions.

In addition to accessing intracellular targets in cell culture, derivinga simple modification that would facilitate oral bioavailability ofpeptidic ligands would be greatly beneficial for therapeuticdevelopment. Intestinal absorption of both fatty acids and biotin isnearly 100% efficient. Zempleni, J. and Mock, D. M. (1999) The AmericanJournal of Clinical Nutrition 69:504. Therefore, Applicants theorizedthat stable peptides with either a biotin or fatty acid conjugationwould exhibit some oral bioavailability. Applicants synthesized aprotease resistant peptide with the sequence MFYAYEYAQWSKK-mod(designated SUPR peptide) (SEQ ID NO: 50), where A is N-methyl alanine,the peptide is cyclized from lysine to the N-terminus, and K(mod) islysine with a modified sidechain including either biotin or palmitoleicacid. Palmitoleic acid is a monounsaturated fatty acid chosen for itsability to bind human serum albumin, and its low melting point allowingfor more favorable solubility of conjugated peptides as compared tounsaturated fatty acid conjugates. This peptide was administered to miceat a dose of 10 mg/kg by oral gavage. At various time points, blood wastaken from the mice by orbital bleeding. After processing, fluorescencewas quantified and compared to the maximum signal obtained by IVinjection of peptide at the same dose. 3 hours after administration,there was a maximum peptide signal corresponding to 9.4% bioavailabilityfor biotinylated peptide and 9.8% bioavailability for peptide conjugatedto fatty acid as can be seen in FIG. 20.

Reacting human insulin with biotin-NHS produced three distinct productsthat can be separated by HPLC. Two peaks showed single modifications andthe third a double modification as determined by MALDI-TOF. Both singlemodifications were examined for in vivo function, but peak 2 was foundto be more efficacious. Chymotrypsin digest and subsequent MALDIanalysis showed that the site of biotinylation was on the N-terminus ofthe B-chain. This agrees with the literature president that modificationat this position does not alter efficacy. Calceti, P. et al. (2004)European Journal of Pharmaceutical Sciences 22:315; and Tuesca, A., etal. (2009) Pharmaceutical Research 26:727. Intravenous injection of thiscompound at a dose of 3.5 nmol/kg showed a similar effect on blood sugaras unmodified insulin both in terms of total intensity of response, andduration of that response (FIG. 21).

Biotinylated insulin and insulin were fed to mice by oral gavage. At adose of 7 nmol/kg, insulin had no effect on blood sugar. Howeverbiotinylated insulin administered orally produced a typical insulinresponse. The longer duration of response is likely due to the kineticsof absorption by the intestinal mucosa.

In addition to biotin, Applicants' data suggests that any highlybioavailable small molecule could deliver similar results. These includeother vitamins, saturated or unsaturated fatty acids, hydrophobic smallmolecules, and saturated and unsaturated hydrocarbons and could be atthe N-terminus of the B-chain or the side chain of Lys. Additionally,other insulin derivatives and analogs such as insulin lispro andinsulinotropic compounds such as GLP-1 analogs could be modified by theinclusion of a biotin for the purpose of blood sugar regulation.

Applicants were also investigating if the significant stability ofcycSUPR would better translate into in vivo stability than the naturalpeptides sequences tested. Previous work showed that sarcosine polymers(N-methyl glycine) showed significant in vivo half-life enhancementswith respect to glycine polymers. It was suspected that this was due toa resistance to renal clearance that the sarcosine conferred on thepeptides. To that end mice were administered peptide conjugated tofluorescein by IV injection into the tail vein. Blood samples were takenby orbital bleeding. Blood peptide concentration was determined bycomparing fluorescence to a 15 minute time point. A dramatic enhancementin the stability of SUPR in vivo was found. CycGIBP showed a half-lifeof 3.1 minutes while cycSUPR peptide had a half-life of 110 minutes, ora roughly 35 fold increase in half-life. The discrepancy in half-lifebetween the serum data and the in vivo data is likely due to renalclearance. Previous work has shown that conjugating a peptide to a smallmolecule that has affinity for serum albumin can significantly lower theclearance rate of a peptide. Albumin, which has a concentration rangingfrom 500-800 μM in blood, has a nanomolar affinity for C-14 to C-18fatty acids. However, initial attempts to synthesize peptide with fattyacid conjugates resulted in molecules with poor solubilities inconditions necessary for in vivo work. A single cis-unsaturation in theC-16 fatty acid palmitoleic acid results in a reduction of melting pointfrom 63° C. to 0° C. Fatty acids with single unsaturations also retainnanomolar binding affinity towards albumin. Applicants found thatpeptides conjugation to palmitoleic acid retained sufficient solubilityin these experiments. Additionally, there was a significant increase inthe in vivo half-life. CycSUPR peptide containing this modification atthe C-terminus exhibited an order of magnitude increase in half-life,1100 minutes, 354 fold higher than cycGIBP as may be noted in FIG. 20.

There are examples of NRPB that are orally bioavailable. Cyclosporin isone such example showing 25% oral bioavailability. However, designedpeptides rarely show any significant oral uptake. One of the mostefficient examples is a stapled helix designed by Walensky et al.(2004), supra. Even in this case, oral availability was limited, lessthan 0.2% uptake. Applicants wanted to determine the efficiency of oraluptake of our peptide in comparison to what has been noted in the field.Applicants examined SUPR peptide with and without the addition of thepalmitoleic acid residue. Applicants also examined SUPR peptidecontaining a C-terminal biotinylation. SUPR peptide without modificationdid not show any detectable oral uptake, as Applicants anticipated.However, both biotin and palmitoleic acid have nearly 100% oralbioavailabilities. The peptides made by the method of this disclosurecan be further modified by conjugation with biotin or a biotin analog,as described in Example 3. Therefore, Applicants postulated thatpeptides conjugated to these molecules would exhibit some oralavailability. In fact, this is precisely what Applicants found asoutlined in FIG. 21. After a single oral administration, there seemed tobe a maximal uptake of biotinylated peptide at 3 hrs. The C-terminalbiotinylation and palmitoleic acid conjugation showed an oral uptakeefficiency of 6.4% and 9.4% respectively, which is well within theacceptable range of therapeutics. Further optimization could likely beachieved by using standard formulation techniques such as themicroencapsulation techniques employed for enhancing cyclosporinedelivery.

Experiment No. 4

The human serum and human microsome stability of the Gαi1 binding SUPRpeptide (MFYAYEYAQWSK (SEQ ID NO: 7)) were compared to the peptidesability to bind to Her-2. Beginning with the human serum analysis,peptides were digested with 95% human serum at 37° C. Analysis wasperformed by HPLC. Any modification to the peptide resulted in a changein retention time, and was subtracted from the sample. Therefore, intactpeptide is only completely unmodified peptide. Digestions of SUPR, HMP,and PMP are show have lives very similar to SUPR. Digestion of PMP2 isin progress. Data is shown in FIG. 23. Without being bound by theory,Applicants theorize that the fitting for HMP may have produced anartificially low half-life. However, all peptides exhibited half-livesof over 100 hrs under stringent digestion conditions.

A similar experiment was performed analyzing protease resistance tocytochrome P450 degradation. Peptide was incubated with human livermicrosomes at 37° C. All peptides were highly protease resistant.However, SUPR was more preotease resistant than the Her-2 bindingpeptides. The selection for SUPR peptide was performed with a morestringent proteolytic pressure. This seems to indicate that cytochromeP450 degradation can be further enhanced during the selection process bydialing up the protease selective pressure step. Data for these peptidesare illustrated in FIG. 24.

Structural Analysis by Circular Dichroism

To show that the structure of the SUPR peptides might contribute tostability, Applicants conducted circular dichroism (“CD”) experiments.Peptide at 50 μM concentration was dissolved in 50 mM potassiumphosphate buffer. CD experiments followed standard protocols, and wereconducted at 20° C. cycGIBP, a cyclic peptide that binds Gαi1 with 2 nMaffinity, seemed to be entirely unstructured (FIG. 25A). All stabilizedpeptides were structured. In the case of SUPR peptide, the compoundseems helical (FIG. 25B). Peptides binding to Gαi1 have sequencesimilarity, and have been shown to be helical in crystal structure data.Therefore this structural motif would be expected in SUPR peptideAnother interesting finding is that SUPR peptide is structured both as alinear and a cyclic peptide (FIGS. 25B and 25D). However, removal of oneor both N-methylations results in a nearly complete loss of structure(FIG. 25D). The Her-2 binding peptide have a CD signal characteristic ofa (3-turn. In fact, PMP's spectra extremely similar to that ofcyclosporine. The data is shown in FIG. 26.

Analyzing the Toxicity of the Stabilized Peptides

An mtt assay was performed to examine the effect of our peptides on cellthat do not overexpress Her-2. Standard protocols were followed.Briefly, 100,000 HEK293T cells were seeded per well in a 96 well plate,and cultured with D10 media. Peptide at various concentrations wasadministered for 36 hr. mtt analysis followed manufacturer'sinstructions The experiment was performed in triplicate. Plotted is theaverage and standard deviation.

There was no apparent toxicity when peptide was administered up to 100μM (FIG. 27). The DMSO concentration of all samples was 1% by volume.Toxicity as measure against liver cells is planned.

Analyzing the Immunogenicity of Stabilized Peptides

Immune response was tested to the repeated administration of our peptidesamples. Two peptides were initially tested: SUPR and cycGIBP. Both ofthese peptides bind to Gαi1 with high affinity. However, only SUPRpeptide has been stabilized.

3 mice per group (C57BL) were tested. 100 ms of peptide was administeredwith Freund's adjuvant to help elicit an immune response. Peptide wasadministered every other week for 8 weeks. At week 10, blood sampleswere taken for analysis.

Analysis followed published protocols. Serum from mice is stored at −80°C. until needed. An ELISA assay was performed using streptavidin coatedplates. Plates were incubated with biotinylated peptide followed byserial dilutions of serum stock (from 1× to 1/2000). After incubationand washing, anti-mouse antibody conjugated with HRP was added. Afterwashing again, substrate solution was added for 15 followed bydeveloping (stop) solution. Absorbance was measured at 450 nM.

As a positive control, the above protocol was followed substitutingbiotinylated mouse antibody for biotinylated peptide. The negativecontrol used no biotinylated product (peptide or antibody). There seemsto be a very low signal for immunogenicity (FIG. 28).

Amino acids are identified by single letter codes or three letter codes.For the sake of clarity, the abbreviations and codes identify thefollowing amino acids.

1-Letter 3-Letter Amino Acid Y Tyr tyrosine G Gly glycine F Phephenylalanine M Met methionine A Ala alanine S Ser serine I Ileisoleucine L Leu leucine T Thr threonine V Val valine P Pro proline KLys lysine H His histidine Q Gln glutamine E Glu glutamic acid W Trptryptophan R Arg arginine D Asp aspartic acid N Asn asparagine C Cyscysteine

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All nucleotide sequencesprovided herein are presented in the 5′ to 3′ direction.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

1-7. (canceled)
 8. A method for generating one or stable, bioavailablepeptide(s), comprising: a. mutating a library of nucleic acids encodingpeptides selected for a pre-determined specificity to incorporate one ormore amino acids that impart stability; b. incorporating one or morestop codons; c. supplementing the library with one or more tRNA chargedwith an amino acid that imparts stability to suppress the stop codon; d.translating one or more of the individual sequences of the nucleiclibrary to one or more peptides; e. cyclizing the one or more peptides;and f. selecting individual peptides for protease resistance wherein theamino acid that imparts stability is selected from the group of: an2-aminoisobutyric acid (AIB), a D-amino acid, a beta (B) amino acid, andan N-methyl amino acid.
 9. The method of claim 8, wherein the nucleicacid library is a DNA library or RNA library.
 10. (canceled)
 11. Themethod of claim 8, further comprising: g. conjugating the peptide to abiotin molecule or biotin analog through a linkage.
 12. The method ofclaim 11, wherein the biotin molecule or biotin analog is one or moreof: is a reducible biotin molecule; a biotin on the side chain of lysineof the peptide; a biotin linked to the peptide through an amide linkageor an ester linkage, a biotin linked to the peptide through a thioesterlinkage or an ester linkage.
 13. (canceled)
 14. The method of claim 8,wherein the peptide selected for a predetermined specificity ispre-selected by a method selected from the group of: specificity againsta cell surface protein, and/or specificity against mammalian cellculture or tissue; and/or specificity against a purified protein target.15-16. (canceled)
 17. The method of claim 14, wherein the purifiedprotein target is a target of a scanning unnatural protease resistantpeptide (“SUPR”).
 18. (canceled)
 19. The method of claim 8, wherein thepeptide selected for a pre-determined specificity is pre-selected for anin vivo therapeutic utility. 20-21. (canceled)
 22. An isolated peptidegenerated according to the method of claim
 8. 23. (canceled)
 24. Thecomposition of claim 64, wherein the carrier is a pharmaceuticallyacceptable carrier. 25-36. (canceled)
 37. A vector comprising anisolated nucleic encoding the isolated peptide of claim
 22. 38. Anisolated host cell comprising an isolated nucleic acid encoding theisolated peptide of claim
 22. 39. A method for producing a non-naturallyoccurring peptide comprising growing one or more an isolated host cellsof claim 38 under conditions that allow expression of the isolatednucleic acid.
 40. The method of claim 39, further comprising isolatingthe non-naturally occurring peptide from the isolated host cells or cellsupernatant. 41-61. (canceled)
 62. The method of claim 8, wherein saidN-methyl amino acid is N-methyl alanine.
 63. The method of claim 8,wherein said in vivo therapeutic utility is selected from the group of:the ability to inhibit the growth or kill a cancer cell, the ability toinhibit or kill a precancerous cell, and to bind to a cell expressing adesired target receptor.
 64. A composition comprising the isolatedpeptide of claim 22 and a carrier.
 65. An isolated peptide librarycomprising a plurality of peptides generated according to the method ofclaim
 8. 66. An isolated nucleic acid library comprising isolatednucleic acids encoding each of a plurality of peptides generatedaccording to the method of claim
 8. 67. The isolated nucleic acidlibrary of claim 66, wherein the nucleic acid library is a DNA libraryor RNA library.